CN115433282A

CN115433282A - anti-CD 3 antibody variant, fusion protein and application

Info

Publication number: CN115433282A
Application number: CN202210629359.XA
Authority: CN
Inventors: 屈向东; 潘琴
Original assignee: Qiyu Biotechnology Shanghai Co ltd
Current assignee: Qiyu Biotechnology Shanghai Co ltd
Priority date: 2021-06-02
Filing date: 2022-05-31
Publication date: 2022-12-06
Also published as: WO2022253248A1; EP4349870A1

Abstract

The invention discloses an anti-CD 3 antibody variant, a fusion protein and application. Variants thereof were obtained by mutating anti-CD 3 antibodies. The fusion protein provided comprises: (1) anti-Tumor Associated Antigen (TAA)/anti-CD 3 bispecific antibody; (2) anti-Tumor Associated Antigen (TAA), anti-CD 3 and multifunctional fusion protein containing IL15/IL15 Ra; (3) anti-Tumor Associated Antigen (TAA) and fusion protein containing IL15/IL15 Ra. Nucleic acid molecules and vectors encoding the antibody molecules are also provided, as well as pharmaceutical uses of the antibody molecules.

Description

anti-CD 3 antibody variant, fusion protein and application

Technical Field

The invention relates to the technical field of biomedicine, in particular to an anti-CD 3 antibody variant, a fusion protein and application.

Background

T lymphocytes (T lymphocytes), abbreviated as T cells, are bone marrow-derived lymphoid stem cells that, after differentiation and maturation in the thymus, are distributed through lymph and blood circulation to immune organs and tissues throughout the body to exert an immune function. T cells are efficient killer cells and can rapidly eliminate virus infected cells and cancer cells. T cell killing requires the formation of an immunological synapse, a process that is highly dependent on the TCR recognition of a complex of MHC molecules on the surface of an antigen presenting cell and the presented antigenic peptide. The activated immunological synapse will release cytotoxins and cytokines for killing. In the course of immune synapse formation, bi-or multi-specific antibody targeting of T cells is limited by the distance between the T cell and the target cell, which mimics immune synapse formation, by targeting CD3 at one end to bridge the TCR receptor of the T cell and at the other end to bridge the target cell by targeting the target cell surface antigen. CD3 molecules are expressed on the surface of all mature T cells, and are non-covalently bound with TCR to form a complete TCR-CD3 complex, which is commonly involved in immune response to antigen stimulation, and is the trigger molecule on the surface of immune effector cells which is most and successfully applied to bispecific antibodies at present. The bispecific antibody targeting CD3 can respectively bind to CD3 on the surface of a T cell and an antigen on the surface of a tumor cell, thereby shortening the distance between a cytotoxic T cell (Tc or CTL) and the tumor cell, directly activating the T cell, inducing the T cell to directly kill the cancer cell, and not relying on the traditional dual activation signal of the T cell.

Among the CD3 antibodies disclosed, OKT3 is an antibody with a milestone impact, which, as the first therapeutic antibody to market, opened the beginning of monoclonal antibody treatment of disease. SP34 is a CD3 antibody that is currently largely published as being able to react with monkey species. In recent years, the scientific community has made a new breakthrough in the field of double antibodies, and thus a new type of CD3 double antibody (CD 3-BsAb) has been developed. At present, more than 100 CD 3-BsAbs with different structures have been successfully developed. The numerous formats determine the properties of the antibody, such as half-life, immunogenicity, type of therapeutic response, ability to penetrate solid tumors, and the like. The Advance Blinatumomab (CD 19xCD3 in BiTE format) obtained FDA approval to market in 2014, and is currently the most successful CD3-BsAb double antibody. The medicine can be used for treating relapsed or refractory Acute Lymphoblastic Leukemia (ALL) patients. Complete or partial remission (CR/PR) can be achieved in more than 40% of patients treated with Blinatumomab compared to standard chemotherapy, with median overall survival being increased by many months. In addition to Blinatumomab, many other CD 3-bsabs are currently undergoing clinical trials, such as CD19, CD20, CD38, BCMA, CD33, and CD123, among others. In addition, patients with Acute Myeloid Leukemia (AML) were treated with Flotetuzumab (CD 123xCD 3-BsAb) by Macrogenetics in a phase 1/2 clinical trial with a total effective rate (ORR) of 30%. Another phase 1/2 clinical trial of Epcotimab (CD 20xCD 3-BsAb) by Abbvie/Genmab showed a Complete Remission (CR) of 44% and a Partial Remission (PR) of 11% in patients with diffuse lymphoid large B-cell tumor (DLBCL), while patients with follicular lymphoma showed 100% remission (PR). Similar excellent results were obtained with several other CD20xCD3 diabodies, for example, REGN1979 (CD 20xCD 3-BsAb) from R.C. in NSG mice better controls tumor growth than Rituximab (CD 20 mAb) from Roche, further demonstrating the efficacy of CD3 diabodies. The recent data for roche CD20xCD 3T cell binding bispecific antibody glofitamab (formerly CD 20-TCB) treatment of relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) patients, the expanded study included over-pretreated and highly refractory DLBCL patients, 58.3% of whom were ineffective for their initial treatment, about one-third (33.1%) of whom had previously received CAR-T cell treatment. Median follow-up at 12.6 months, according to the evaluation of the independent review board (IRC), 39.4% of patients (61/155) reached CR (primary efficacy endpoint), half of patients (51.6%; 80/155) reached overall remission (proportion of patients who had partial remission [ PR ] + complete remission [ CR ]; secondary efficacy endpoint), and in terms of safety, cytokine Release Syndrome (CRs) was the most common adverse event occurring in 63.0% of patients. CRS events were predictable, generally low grade (mainly grade 1 [47.4% ] or grade 2 [11.7% ]), occurring at the initial dose, with only one patient stopping glofitamab treatment with CRS. The incidence of CRS of grade 3 and above was low (3.9%), with no grade 5 events. The glofitamab has a novel structural pattern of "2.

Blinatumomab of ann corporation showed cytokine storm (CRS) as one of its most important toxic side effects in clinical trials. CD19 expressing tumor cells, in the same environment as a large number of healthy B and T cells, cause CD3-BsAb mediated T cell activation to proceed abruptly, thereby releasing inflammatory cytokines such as IFN-. Gamma., IL-6 and TNF-. Alpha.excessively, and finally causing symptoms ranging from fever to multiple organ failure. However, CRS is not a unique toxic side effect of Blinatumomab. Indeed, CRS is frequently present in most CD3-BsAb and CAR-T therapies. In a humanized mouse model, it was shown that the main mediator of CRS induction by CD3-BsAb is TNF- α produced by activated T cells, resulting in massive secretion of inflammatory cytokines by monocytes. It was found that blocking upstream TNF- α and downstream IL-1 β or IL-6 could be effective in mitigating CRS. In addition, several preclinical studies in mouse and monkey models have shown that by reducing the affinity of CD3 (i.e., using "weaker" CD 3), the extent of the cytokine storm can be reduced.

Interleukin-15 (IL-15) is a cytokine of 14-15kDa in length and is important for NK cell, NKT cell and memory CD8+ T cell function. IL-15 is transduced to target cells together by binding to its receptor IL-15Ra to produce a complex IL-15 superagonist (IL-15 SA) with a very high biological potency. IL-15SA is intenseCells, particularly NK/T cells, that respond to expression of IL-15R are activated, thereby promoting anti-tumor and anti-viral functions. IL-15 has a broad immunomodulatory role and can be involved in the regulation of survival, proliferation and function of T cells, particularly NK cells, memory CD8+ T cells. IL-15 has a structure very similar to IL-2, and belongs to the spiral cytokine family. IL-15 heterotrimeric receptors share IL-2R/IL-15R β (CD 122) and a common γ c chain (CD 132) with the IL-2 receptor, and due to these common receptor components and the common JAK 1/JAK 3/STAT 5 signaling pathway, IL-15 has the same functions as IL-2, including stimulation of T cell proliferation, production of cytotoxic T lymphocytes, stimulation of immunoglobulin synthesis by B cells, and production and sustained survival of NK cells. In many adaptive immune responses, IL-2 and IL-15 have unique and often competitive effects. The following four points are mainly provided: 1. IL-2 can regulate the activation of Tregs cells, while IL-15 cannot. 2. IL-2 may inhibit T cell responses by activating induced cell death of CD8+ effector T cells. 3. IL-15 plays an essential role in the differentiation of NK, effector CD8+ and memory phenotype CD8+ T cells. 4. Preclinical studies have shown that IL-15 is not as toxic as IL-2, and that little vascular capillary leakage is observed with IL-15 compared to IL-2. These factors make IL-15 a more potent cytokine in tumor immunization strategies. Currently, a plurality of IL-15 target products are still in clinical trial, wherein the most notable is IL-15 super agonist N-803, which is a fusion protein co-expressed by IL-15 protein N72D after mutation in combination with IL-15Ra and IgG1Fc, 23.5.2022.ImmunityBio submitted IL-15 super agonist N-803 to FDA for combined treatment with BCG (BCG) for non-muscle invasive bladder cancer (NMIBC) to which BCG does not respond. Clinical data published in 2021 shows that the combination treatment effect of N-803+ BCG is remarkable, the CR rate reaches 71% (59/83), and the average CR maintenance time is 24.1 months. A phase 1 clinical experiment shows that the combination of Nivolumab (PD-1 antibody, opdivo) and N-803 in treating metastatic non-small cell lung cancer can obviously prolong the long-term survival of patients. Gene tack and Xencor company collaboratively develop IL-15 cytokine

Is currently underway

Combined with a Tecentriq clinical study. The SHR-1501 (IL-15 fusion protein) of Hengrui medicine was approved to carry out SHR-1501 clinical trial 5, 14 months and 2019. In addition, SHR-1316 (PD-L1 mAb drug) will be used in combination with SHR-1501 to treat patients with advanced malignant tumors that fail.

The development requirement of CD3 bispecific antibodies for therapy is still not met, and development and research are needed to solve the challenges faced in solid tumors and improve the safety of CD3-BsAb double antibodies.

Disclosure of Invention

A first object of the present invention is to provide an anti-CD 3 antibody variant, said anti-CD 3 antibody comprising: a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises: VHCDR1, VHCDR2, VHCDR3; the light chain variable region comprises: VLCDR1, VLCDR2, VLCDR3; the amino acid sequence of the anti-CD 3 antibody comprises SEQ ID NO: VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of 29-34; the mutation sites of the anti-CD 3 antibody variant comprise any 1 or more than 2 of the following sites: SEQ ID NO:30, the tenth position of the sequence shown in SEQ ID NO:31, the second and seventh sites of the sequence shown in SEQ ID NO:33, the third and fourth sites of the sequence shown in SEQ ID NO:34, the fourth, fifth, sixth and seventh sites of the sequence shown in the specification.

Preferably, the mutation is a substitution of an amino acid.

Preferably, the mutation site of the variant includes any 1 or 2 or more of the following sites: SEQ ID NO:30 is substituted with E at the tenth position of the sequence shown in SEQ ID NO:31 wherein G at the second position is substituted by S, S at the seventh position is substituted by G, SEQ ID NO:33 is substituted W for N at the third position, L for K at the fourth position, SEQ ID NO:34 is substituted by N or R at the fourth position, K at the fifth position, G at the sixth position and G at the seventh position.

Preferably, the amino acid sequence of VHCDR2 is as shown in SEQ ID NO 35; or the amino acid sequence of VHCDR3 is shown as SEQ ID NO. 36; or the amino acid sequence of VLCDR2 is shown as SEQ ID NO 37 or 38; alternatively, the amino acid sequence of VLCDR3 is set forth in SEQ ID NO 39, 40 or 41.

Preferably, the amino acid sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 are as shown in any one of the following sets of sequences:

(1)SEQ ID NO：29、35、31、32、37、39；

(2)SEQ ID NO：29、35、36、32、37、40；

(3)SEQ ID NO：29、35、36、32、38、41。

preferably, the heavy chain variable region and/or the light chain variable region of the antibody variant is selected from SEQ ID NO: 1.2 or 3, or at least 90% sequence identity thereto.

Preferably, the heavy chain variable region and the light chain variable region have a mutation for disulfide bond formation between them at a site including any one or two or more of the following combinations, the mutation sites being numbered according to EU, the heavy chain variable region being represented by VH, and the light chain variable region being represented by VL:

preferably, the anti-CD 3 antibody variant further comprises: a heavy chain constant region selected from human IgG1, igG2, igG3, or IgG4, or a variant thereof; and a light chain constant region selected from a human kappa, lambda chain or variant thereof; the heavy chain constant region comprises: an Fc fragment or variant thereof.

Preferably, the antibody variant is a scFv comprising a heavy chain variable region, a light chain variable region, and a linker joining the heavy chain variable region and the light chain variable region, the amino acid sequence of the linker preferably being several GGGGS repeats, more preferably 3 GGGGS repeats.

The invention also aims to provide application of the anti-CD 3 antibody variant in preparing a medicament for inhibiting or treating cancer.

Preferably, the cancer is selected from or occurs at: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphatic cancer, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.

It is another object of the invention to provide a nucleic acid molecule encoding said anti-CD 3 antibody variant.

Another object of the present invention is to provide an expression vector comprising the above-described nucleic acid molecule.

It is another object of the present invention to provide an anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule, which is in a heterodimeric form, comprising a first monomer and a second monomer;

the first monomer includes: (a) an Fd fragment; (b) a light chain fragment and a first Fc chain; the second monomer includes: (a) an Fd fragment; (b) a light chain fragment, an anti-CD 3 antibody fragment, and a second Fc chain; the light chain fragment comprises a VL domain and a CL domain; the Fd fragment comprises a VH domain and a CH1 domain; pairing the light chain fragment with the Fd fragment in the first monomer or the second monomer to form an anti-TAA Fab domain; wherein the light chain fragment of the first monomer is fused to the first Fc chain; the N-terminus of the anti-CD 3 antibody fragment is fused to the light chain fragment of the second monomer and the C-terminus is fused to the second Fc chain;

alternatively, the first monomer comprises: (a) an Fd fragment; (b) a light chain fragment, a cytokine domain, a first Fc chain; the second monomer includes: (a) an Fd fragment; (b) A light chain fragment, an anti-CD 3 antibody fragment, and a second Fc chain; the cytokine functional region comprises IL-15 and IL-15Ra; wherein the cytokine functional region is fused to the light chain fragment of the first monomer at the N-terminus and to the first Fc chain at the C-terminus; the N-terminus of the anti-CD 3 antibody fragment is fused to the light chain fragment of the second monomer and the C-terminus is fused to the second Fc chain;

alternatively, the first monomer comprises: an anti-TAA antibody fragment, a cytokine functional region, a first Fc chain; the second monomer includes: an anti-TAA antibody fragment, an anti-CD 3 antibody fragment, and a second Fc chain; wherein the cytokine domain is fused to the anti-TAA antibody fragment of the first monomer at the N-terminus and to the first Fc chain at the C-terminus; the N-terminus of the anti-CD 3 antibody fragment is fused to the anti-TAA antibody fragment of the second monomer and the C-terminus is fused to the second Fc chain;

the first Fc chain is interchangeable with the second Fc chain.

Preferably, the anti-TAA antibody fragment is in scFv format comprising: a heavy chain variable region (VH), a light chain variable region (VL), and a linker linking the heavy chain variable region and the light chain variable region; the amino acid fusion sequence of the anti-TAA antibody fragment is "VH to VL", or "VL to VH", from the N-terminus to the C-terminus of the peptide chain; "-" indicates a connection fragment; alternatively, the anti-CD 3 antibody fragment is in the form of an scFv comprising: a heavy chain variable region (VH), a light chain variable region (VL), and a linker linking the heavy chain variable region and the light chain variable region; the fusion sequence of the amino acids of the anti-CD 3 antibody fragment is "VH to VL", or "VL to VH", from the N-terminus to the C-terminus of the peptide chain; "-" indicates a connecting fragment; or, from the N-terminal to the C-terminal of the peptide chain, the fusion sequence of the amino acid fragments of the cytokine functional region is 'IL-15 to IL-15 Ra', or 'IL-15 Ra to IL-15'; "-" indicates a connection fragment; preferably, the sequences of the IL-15 to IL-15Ra are shown in SEQ ID NO. 42, and the sequences of the IL-15Ra to IL-15 are shown in SEQ ID NO. 43.

Preferably, the amino acid sequence of the anti-CD 3 antibody fragment is selected from the group consisting of a specific antibody that binds CD3, an antibody fragment, a single domain antibody or a humanized form thereof; preferably, the amino acid sequence of the anti-CD 3 antibody fragment is selected from SP34, OKT3, UCTH1 or derivatives thereof.

Preferably, the anti-CD 3 antibody fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region of the anti-CD 3 antibody fragment comprises: VHCDR1, VHCDR2, VHCDR3; the light chain variable region of the anti-CD 3 antibody fragment comprises: VLCDR1, VLCDR2, VLCDR3; the amino acid sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are represented by any one of the sets of sequences in the anti-CD 3 antibody variant of claim 5.

Preferably, IL-15Ra and IL-15 form an IL-15/IL-15Ra complex; IL-15 includes: IL-15 and mutations, truncations and various derivatives thereof that bind IL-15Ra; IL-15Ra includes: IL-15Ra and mutations, truncations and various derivatives thereof that bind IL-15; preferably, the IL-15/IL-15Ra complex includes, but is not limited to, the mutation pattern shown in any combination of the following, counting from the first amino acid of the amino acid sequence of IL-15 or IL-15Ra to position 1; preferably, the parent sequence of the IL-15 is shown as SEQ ID NO: 44; the parent sequence of the IL-15Ra is shown as SEQ ID NO: 45:

combination (I)	IL15	IL15Ra
				1	wt	D96
2	wt	D96/P97
			3	wt	D96/P97/A98
4	E87C	D96/C97
			5	E87C	D96/P97/C98
6	E87C	D96/C97/A98
			7	V49C	S40C
8	L52C	S40C
			9	E89C	K34C
10	Q48C	G38C
			11	E53C	L42C
12	C42S	A37C
			13	L45C	G38C
14	L45C	A37C

。

Preferably, the IL-15 includes, but is not limited to, the mutation pattern shown by any one combination of the following, counting from the first amino acid of the amino acid sequence of IL-15 to position 1; preferably, the parent sequence of the IL-15 is shown in SEQ ID NO: 44:

combination (I)	IL15 mutations
		1	N1D
2	N4D
		3	D8N
4	D30N
		5	D61N
6	E64Q
		7	N65D
8	Q108E
		9	N1D/D61N
10	N1D/E64Q
		11	N4D/D61N
12	N4D/E64Q
		13	D8N/D61N
14	D8N/E64Q
		15	D61N/E64Q
16	E64Q/Q108E
		17	N1D/N4D/D8N
18	D61N/E64Q/N65D
		19	N1D/D61N/E64Q/Q108E
20	N4D/D61N/E64Q/Q108E

。

Preferably, the TAA is selected from the group consisting of CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276, PD-1, CEA, epcam, trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR VIII, C-MET, HER2, FGFR3, PSMA, PSCA, ephA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, or Glypican-3.

Preferably, the first and second Fc chains polymerize to form an Fc fragment; the Fc segment is selected from Human IgG1Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc or variants thereof, preferably from IgG1Fc or Human IgG4 Fc or variants thereof; the protein molecule is in the form of an Fc heterodimer; preferably, the Fc heterodimer comprises, but is not limited to, a combination of the following mutations, counted according to EU:

preferably, the protein molecule comprises an Fc fragment, which selects against immune effector functions, including but not limited to the following combinations of mutations, the following mutations being counted according to EU:

preferably, the anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule is obtained by fusing amino acid fragments shown in any group of sequences as follows:

(1)SEQ ID NO:05、SEQ ID NO:06、SEQ ID NO:07；

(2)SEQ ID NO:08、SEQ ID NO:06、SEQ ID NO:07；

(3)SEQ ID NO:09、SEQ ID NO:06、SEQ ID NO:07；

(4)SEQ ID NO:10、SEQ ID NO:06、SEQ ID NO:07；

(5)SEQ ID NO:05、SEQ ID NO:17、SEQ ID NO:07；

(6)SEQ ID NO:01、SEQ ID NO:18、SEQ ID NO:07；

(7)SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:07；

(8)SEQ ID NO:19、SEQ ID NO:18、SEQ ID NO:07；

(9)SEQ ID NO:20、SEQ ID NO:21；

(10)SEQ ID NO:22、SEQ ID NO:21；

(11)SEQ ID NO:20、SEQ ID NO:23；

(12)SEQ ID NO:22、SEQ ID NO:23；

(13)SEQ ID NO:24、SEQ ID NO:25；

(14)SEQ ID NO:26、SEQ ID NO:25；

(15)SEQ ID NO:24、SEQ ID NO:27；

(16)SEQ ID NO:26、SEQ ID NO:27。

another objective of the invention is to provide a nucleic acid molecule encoding the anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule.

Another object of the present invention is to provide the use of the anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule in the preparation of a medicament for inhibiting or treating cancer, wherein the cancer comprises or occurs at the following sites: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphatic cancer, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.

Another objective of the invention is to provide an anti-Tumor Associated Antigen (TAA) and a fusion protein containing IL-15/IL-15Ra, which comprises two polypeptide chains, wherein any one polypeptide chain comprises: anti-TAA antibody fragments, cytokine functional regions, fc fragments; the anti-TAA antibody fragment is in scFv format; the cytokine functional region comprises IL-15 and IL-15Ra; the N-terminus of the cytokine domain is fused to the anti-TAA antibody fragment and the C-terminus is fused to the Fc fragment.

Preferably, the anti-Tumor Associated Antigen (TAA) and the fusion protein containing IL-15/IL-15Ra form IL-15/IL-15Ra complex with IL-15; the IL-15 includes: IL-15 and its mutations, truncations and various derivatives capable of binding IL-15Ra; IL-15Ra includes: IL-15Ra and mutations, truncations and various derivatives thereof capable of binding IL-15; preferably, the IL-15/IL-15Ra complex includes, but is not limited to, the mutation pattern shown in any combination of the following, counting from the first amino acid of the amino acid sequence of IL-15 or IL-15Ra to position 1; preferably, the parent sequence of the IL-15 is shown as SEQ ID NO: 44; the parent sequence of the IL-15Ra is shown as SEQ ID NO: 45:

combination of	IL15	IL15Ra
				1	wt	D96
2	wt	D96/P97
			3	wt	D96/P97/A98
4	E87C	D96/C97
			5	E87C	D96/P97/C98
6	E87C	D96/C97/A98
			7	V49C	S40C
8	L52C	S40C
			9	E89C	K34C
10	Q48C	G38C
			11	E53C	L42C
12	C42S	A37C
			13	L45C	G38C
14	L45C	A37C

。

Preferably, the anti-Tumor Associated Antigen (TAA) and the fusion protein containing IL-15/IL-15Ra have the IL-15 including but not limited to the mutation mode shown by any combination of the following, and the counting mode is counted as the 1 st position according to the first amino acid of the amino acid sequence of the IL-15; preferably, the parent sequence of the IL-15 is shown in SEQ ID NO: 44:

combination of	IL15 mutations
		1	N1D
2	N4D
		3	D8N
4	D30N
		5	D61N
6	E64Q
		7	N65D
8	Q108E
		9	N1D/D61N
10	N1D/E64Q
		11	N4D/D61N
12	N4D/E64Q
		13	D8N/D61N
14	D8N/E64Q
		15	D61N/E64Q
16	E64Q/Q108E
		17	N1D/N4D/D8N
18	D61N/E64Q/N65D
		19	N1D/D61N/E64Q/Q108E
20	N4D/D61N/E64Q/Q108E

。

Preferably, the anti-Tumor Associated Antigen (TAA) and the fusion protein containing IL-15/IL-15Ra comprise an Fc segment which selects to eliminate immune effector functions, including but not limited to the following combinations of mutations, the following mutations are counted according to EU: :

preferably, the amino acid sequence of any one of the polypeptide chains of the anti-Tumor Associated Antigen (TAA) and the fusion protein containing IL-15/IL-15Ra is shown in SEQ ID NO 28, or has at least 90% sequence identity with the polypeptide chain.

Another objective of the invention is to provide a nucleic acid molecule encoding the anti-Tumor Associated Antigen (TAA) and a fusion protein containing IL-15/IL-15 Ra.

Another objective of the invention is to provide an application of the anti-Tumor Associated Antigen (TAA) and the fusion protein containing IL-15/IL-15Ra in preparing a medicament for inhibiting or treating cancers, wherein the cancers comprise the following cancers or occur in the following parts: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphatic cancer, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention mutates the existing anti-CD 3 antibody to obtain a new anti-CD 3 antibody variant.

(2) The invention also provides antibody molecules with novel structures, which have better biological activity.

Drawings

FIG. 1 is a diagram showing FACS detection of the binding of CD3 antibody SP34 and its mutant to Jurkat cells.

FIG. 2 shows the molecular pattern design of anti-TAA (tumor associated antigen) and anti-CD 3 bispecific antibodies.

FIG. 3 shows the molecular design of QP 372337241461.

FIG. 4 shows the molecular design of QP 374437481461.

FIGS. 5 and 6 are graphs showing the results of FACS detection of the binding of the equal molecular of CLDN18.2/CD3 double antibody to CLDN18.2.

FIGS. 7 and 8 are graphs showing the results of FACS detection of binding of molecules such as CLDN18.2/CD3 diabody to CD3.

FIGS. 9 and 10 are graphs showing the results of killing human gastric cancer cells NUGC4-CLDN18.2 cells by PBMC mediated by antibodies such as CLDN18.2/CD 3.

FIG. 11 is a graph of the results of molecule-mediated PBMC killing of human lung cancer cells HCC827-CLDN18.2 cells by antibodies to CLDN18.2/CD 3.

FIG. 12 is a graph of the inhibition of tumor growth by a CLDN18.2/CD3 dual antibody molecule in an in vivo animal model of CD3EGD HuGEMM mice subcutaneously transplanted with colorectal cancer cells MC 38-hCLDN18.2.

FIG. 13 is a graph showing the body weight change of mice in each group after the administration of the in vivo animal model of CD3EGD HuGEMM mice subcutaneously transplanted with colorectal cancer cells MC 38-hLDN18.2.

FIGS. 14 and 15 are graphs showing the inhibition of tumor growth by a CLDN18.2/CD3 dual antibody molecule in a PBMC humanized NOG mouse X-CLDN18.2/MIA PaCa-2 subcutaneous transplantation tumor model.

FIGS. 16-19 are block diagrams of the structural formats of a type of anti-TAA/CD 3 and IL-15/IL-15 Ra-containing multifunctional fusion protein.

FIGS. 20 to 27 are structural format schemes of another class of anti-TAA/CD 3 and IL15/IL15 Ra-containing multifunctional fusion proteins.

FIG. 28 is a structural form diagram of TAA/IL15 bifunctional fusion proteins activated by tumor-targeted T cells/NK cells.

FIG. 29 is a map of FACS detection of CLDN18.2/CD3/IL15 multifunctional fusion protein binding to CLDN18.2.

FIG. 30 is a graph showing the results of FACS detection of CLDN18.2/CD3/IL15 and CLDN18.2/IL15 fusion proteins binding to CLDN18.2.

FIG. 31 is a graph showing the results of FACS detection of binding of the CLDN18.2/CD3/IL15 multifunctional fusion protein to CD3.

FIG. 32 is a graph showing the results of FACS detection of binding of CLDN18.2/CD3/IL15 and CLDN18.2/IL15 fusion proteins to CD3.

FIG. 33, FIG. 34, FIG. 35 are graphs showing the results of Mo7e cell proliferation assay for detecting the activity of multifunctional fusion protein IL 15.

FIGS. 36 and 37 are graphs showing the results of killing human gastric cancer cells NUGC4-CLDN18.2 by PBMC mediated by CLDN18.2/CD3/IL 15.

FIG. 38 is a chart of CLDN18.2/CD3/IL15 mediated killing of human lung cancer cells HCC827-CLDN18.2 cells by PBMC.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.

The experimental method without specifying the specific conditions in the experiment is generally performed under the conventional conditions or the conditions recommended by the manufacturers of the raw materials or the commercial products. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Herein, "/" means "and". For example, an anti-Tumor Associated Antigen (TAA)/anti-CD 3 bispecific antibody refers to an antibody that targets both TAA and CD3.

The term "fusion" refers to fusion by peptide bonds, either directly or via linking segments joining components, or by intermolecular interactions. In a single peptide chain, fusion means linkage by peptide bond directly or via a linking fragment. "multifunctional fusion protein" refers to a protein comprising two or more antigen binding domains capable of binding two or more different epitopes (e.g., two, three or more different epitopes), multifunctional fusion proteins may further comprise cytokines (e.g., IL-15 Ra), and the like. "fusion position" refers to the position of a functional region or domain in a peptide chain, indicating the order of attachment of the functional fragments on the peptide chain.

The term "polypeptide" refers to a chain of amino acids of any length, including proteins and fragments thereof. The present invention discloses polypeptides as sequences of amino acid residues. Those sequences are written left to right in the amino-to carboxy-terminal direction. According to standard nomenclature, amino acid residue sequences are designated by three-letter or one-letter codes as follows: alanine (Ala, a), arginine (Arg, R), aspartylamine ((Asn, N), aspartic acid (Asp, D), hemisarcosine (Cys, C), glutamineamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (I1E, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

The term "single-chain" refers to a molecule comprising amino acids linearly linked by peptide bonds.

The term "variant" or "mutant" refers to a polypeptide or polynucleotide that differs from the amino acid or nucleotide sequence involved but retains essential properties. Typically, the differences between variants or between a variant and the parent antibody are limited and the amino acid sequences are very similar overall. In the present specification, an antibody or an antibody fragment before mutation is referred to as a parent antibody, and an antibody or an antibody fragment after mutation is referred to as a variant. The variant still has antigen binding activity.

The term "antibody" (Ab) refers to an immunoglobulin molecule (Ig) comprising at least one antigen binding site and capable of specifically binding an antigen.

The term "antigen" is a substance that induces an immune response in the body and binds specifically to an antibody. Binding of an antibody to an antigen is mediated by interactions formed between the two, including hydrogen bonds, van der waals forces, ionic bonds, and hydrophobic bonds. The region of the antigen surface to which the antibody binds is an "antigenic determinant" or "epitope", and in general, there are multiple determinants per antigen.

The term "antibody" as referred to herein is understood in its broadest sense and encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies (e.g., bispecific antibodies) comprising at least two different antigen binding domains. Antibodies also include murine, humanized, chimeric, human, and other sources of antibodies. The antibodies of the invention may be derived from any animal including, but not limited to, immunoglobulin molecules from humans, non-human primates, mice, rats, cows, horses, chickens, camels, alpacas, and the like. Antibodies may contain additional alterations such as unnatural amino acids, fc effector function mutations and glycosylation site mutations. Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of the antibodies, and immunoglobulin molecules comprising any other modification to the antigen recognition site, so long as the antibodies exhibit the desired biological activity. In other words, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen binding domain.

The basic structure of an antibody is a Y-shaped monomer consisting of two identical heavy chains (H) and two identical light chains (L) linked by disulfide bonds. Each chain is composed of 2-5 domains (also called functional regions) with similar sequences but different functions, each of which contains about 110 amino acids. The amino acid sequences of the light chain and the heavy chain in the antibody molecule near the N end are greatly changed, and the formed structural domain is called a variable region (V region); the region in which the amino acid sequence is relatively constant near the C-terminus is called a constant region (C region).

The V regions of the heavy and light chains are referred to as VH and VL, respectively, which are highly variable in their amino acid composition and arrangement of 3 regions, each referred to as hypervariable regions (HVRs); this region forms a spatial conformation complementary to the epitope of the antigen, also known as the Complementarity Determining Region (CDR). The 3 CDRs of VH are respectively represented by VHCDR1, VHCDR2 and VHCDR3, and the 3 CDRs of VL are respectively represented by VLCDR1, VLCDR2 and VLCDR 3. The VH and VL together constitute 6 CDRs which constitute an antigen-binding site. The diversity of amino acids in the CDR regions is the molecular basis for the specific binding of antibodies to a large number of different antigens. The V region is called a Framework Region (FR) in which the amino acid composition and arrangement sequence other than CDRs are not greatly changed. VH and VL each have 4 framework (or framework), FR1, FR2, FR3, FR4. Each VH and VL consists of three CDRs and four FRs, arranged in the order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Human immunoglobulins can be classified into 5 classes according to the amino acid sequence of the antibody heavy chain constant region: igM, igG, igA, igD, igE. It can be further divided into different subclasses (isotypes), e.g., human IgG can be divided into IgG1, igG2, igG3, igG4; igA can be classified into IgA1 and IgA2. Subclasses of IgM, igD and IgE have not been found. Light chains can be classified into kappa and lambda chains, depending on their amino acid sequence. The antibodies of the invention may be of any class (e.g., igM, igG, igA, igD, igE) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, igA 2).

The constant regions of the heavy and light chains are called CH and CL, respectively. The heavy chain constant region of IgG, igA and IgD has three structural domains of CH1, CH2 and CH3, and the heavy chain constant region of IgM and IgE has four structural domains of CH1, CH2, CH3 and CH4.

The hinge region (hinge region) is located between CH1 and CH2, contains abundant proline, is easy to stretch and bend, can change the distance between two arms of the Y shape, and is beneficial to simultaneously combining antigen epitopes.

The term "Fab fragment" is an antigen-binding fragment (Fab), meaning an antibody fragment consisting of the VL, VH, CL and CH1 domains, which binds (monovalent) to a single epitope. It is known to those skilled in the art that under certain conditions, certain portions of the antibody molecule chain are susceptible to hydrolysis by proteolytic enzymes into various fragments. Papain hydrolyzes the antibody molecule from the near N-terminus of the hinge region into 2 identical antigen binding fragments (Fab) and one crystallizable fragment (Fc).

The term "Fd fragment" means an antibody fragment consisting of VH and CH1 domains.

The terms "Fc", "Fc fragment", "Fc domain" refer to a fragment of a crystallizable antibody that has no antigen binding activity and is the site of interaction of the antibody with effector molecules or cell surface Fc receptors (fcrs). Fc fragments bind to cells with corresponding Fc receptors on their surface, resulting in different biological effects. In the ADCC effect (antibody-dependent cell-mediated cytotoxicity), the Fab fragment of the antibody binds to an epitope of a virus-infected cell or a tumor cell, and the Fc fragment thereof binds to the FcR on the surface of a killer cell (NK cell, macrophage, etc.) to mediate direct killing of the killer cell to a target cell. The Fc fragment comprises the constant region polypeptides of the antibody other than the heavy chain constant region CH1, namely, the human immunoglobulins IgA, igD, two constant region domains CH2 and CH3 at the carboxy terminus of the IgG heavy chain constant region, and the three constant region domains CH2, CH3 and CH4 at the carboxy terminus of the human immunoglobulin IgE and IgM heavy chain constant regions. The Fc fragment is often selected from Human IgG1Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc or variants thereof, preferably from IgG1Fc, or Human IgG4 Fc or variants thereof

The Fc may be composed of two chains, and the two chains of the Fc fragment are described herein as a first Fc chain and a second Fc chain, which may be mutated, respectively, and the present invention is not particularly limited. An Fc fragment can also refer to a single polypeptide chain in an Fc domain. The Fc portion of the antibody may be selected to eliminate immune effector functions, including but not limited to the following combinations of mutations (as counted according to EU).

Space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions, and the like can be formed between the mutationally designed Fc variants. The interaction between Fc variants contributes to the formation of stable heterodimers. Preferred mutation designs are those in the "Knob-in-hole" format.

scFv (single chain antibody fragment), or single chain antibody, is formed by connecting an antibody heavy chain variable region and a light chain variable region through a linker. "linker fragments" also link IL-15 to IL-15Ra, VH to VL of CD3 antibodies, and ensure proper folding and peptide stability of the protein. The "linker fragment" is preferably (GGGGS) n, where n may be 0, 1, 2, 3, 4, 5 or more. If the sequence of the connecting fragment is too short, the folding of the higher-order structures of the two proteins can be influenced, so that the two proteins can interfere with each other; if the linker sequence is too long, problems with immunogenicity are involved, since the linker sequence itself is a new antigen.

Tumor-associated antigen (TAA), refers to antigenic molecules present on tumor cells or normal cells, including: embryonic proteins, glycoprotein antigens, squamous cell antigens, and the like, are commonly used for clinical diagnosis of tumors. Tumor-associated antigens are not specific to tumor cells, are synthesized in minute quantities by normal cells, and are highly expressed when tumor cells proliferate, and are therefore referred to as "associated antigens". The tumor associated antigen may be, for example, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276, PD-1, CEA, epcam, trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR VIII, C-MET, HER2, FGFR3, PSMA, PSCA, ephA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB or Glypican-3.

The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids

The terms "IL-15", "IL-15Ra" may be mutants or fragments thereof.

The terms "first" and "second" are used for descriptive purposes only and do not indicate or imply a degree of importance.

The present invention uses the existing CD3 antibody SP34 as a parent antibody, and mutates the variable region of the parent antibody to obtain the CD3 antibody variant. The invention also provides a plurality of protein molecules aiming at different targets and having different configurations.

The following sequences can be used for "IL-15 to IL15 Ra":

SEQ ID NO:42

the following sequences can be used for "IL15Ra to IL-15":

SEQ ID NO:43

1. anti-CD 3 antibody variant acquisition and format design of anti-TAA (tumor associated antigen)/CD 3 bispecific antibody

Example 1: anti-CD 3 antibody variant acquisition, molecular cloning, transient expression, and protein purification

1. Molecular cloning: humanized anti-CD 3 antibody SP34 (Wileman et al, 1990, US 8236308) is used as a female antibody, CDR mutation is carried out, and an antibody with high stability is screened to obtain 3 variants. The light and heavy chain variable regions of the CD3 parent antibody SP34 and the variant thereof are constructed into a single-chain antibody VH-VL through a connecting sequence, the C end of the single-chain antibody VH-VL is fused with an FC segment of human IgG1, and the single-chain antibody VH-VL is constructed and loaded into an eukaryotic expression vector pQD through molecular cloning. The clone numbers and protein sequence numbers are shown in the following table:

table 1: CD3 antibody SP34 and mutant antibody numbering thereof

The amino acid sequence is shown as follows:

SEQ ID NO:01QD3685

SEQ ID NO:02QD3689

SEQ ID NO:03QD3690

SEQ ID NO:04QD3679(hSP34 VH-VL-FC)

note: the underlined amino acids represent the heavy chain variable region of the antibody, the wavy line portion represents the light chain variable region, and the italic portion between the heavy chain variable region and the light chain variable region represents the linker sequence. The variable region has the amino acid sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the portions with bold and bottom bold lines show HCDR1, HCDR3, LCDR1, LCDR2, LCDR3, respectively. The parent antibody and its mutant CDR sequence comparisons are shown in the following table:

table 2: comparison of CDR sequences of parent antibodies and mutants thereof

2. Protein expression: 293E cells adjusted to a cell density of 1X 10 ⁶ And (4) the concentration is/ml. The plasmid and PEI as transfection reagent were prepared, and the amount of the plasmid to be transfected was 100. Mu.g/100 ml of cells, and usedThe mass ratio of PEI to plasmid was 2. The plasmid and PEI are mixed evenly and kept stand for 15min. Slowly adding the plasmid and PEI mixture to 293E cells, charging 8% CO ₂ Cultured on a shaker at 120rpm and 37 ℃. On the fifth to sixth day of transfection, cell supernatants were collected and purified by centrifugation at 4700rpm for 20min in a horizontal centrifuge.

3. Protein purification:

protein affinity chromatography: the cell culture fluid is centrifuged at high speed, and the supernatant is taken out and subjected to affinity chromatography by using a GE Protein A chromatographic column. The chromatography uses an equilibrium buffer solution of 1 XPBS (pH7.4), after cell supernatant is combined, the cell supernatant is washed by PBS until ultraviolet rays return to a baseline, then 0.1M glycine (pH3.0) is used as an elution buffer solution to elute the target protein, and Tris is used for adjusting the pH value to be neutral for storage; protein size exclusion chromatography: the product obtained by ion exchange is concentrated by ultrafiltration and then subjected to size exclusion chromatography, for example separation on GE Superdex200 gel, to remove possible aggregates and other components and obtain the desired product of high purity. The purity of the protein obtained can be analyzed by SDS-PAGE and SEC-HPLC detection. Protein concentration was determined by uv spectrophotometry.

The yield and purity of cell expressed protein are shown in the table below, wherein QP3679, namely the maternal humanized anti-CD 3 antibody SP34, has the yield of 68.64mg/L, and anti-CD 3 antibody mutants show the expression yield higher than that of the maternal antibody QP3679, so that the mutant yield is higher, the possible protein is more stable, and the protein purity detected by SEC-HPLC is good.

Table 3: transient yield and protein purity of SP34 antibody and variant thereof

Example 2: FACS detection of anti-CD 3 antibody variants binding to naturally expressing CD3 Jurkat cells

FACS detection of anti-CD 3 antibody binding activity: human lymphocytic leukemia cells (Jurkat cells) express native CD3 and binding of anti-CD 3 antibodies and mutants thereof to Jurkat cells is detected by FACS (flow cytometry). 1E 5/well Jurkat cells are planted in a U-shaped 96-well plate, washed once with ice PBS and centrifuged for 3min at 1200 rpm; after washing, 200. Mu.L/well of 3% FBS/PBS blocking solution was added, and incubated on ice for 1h. After sealing, centrifuging at 1200rpm for 3min, throwing off the supernatant, incubating samples with different concentrations, incubating for 2h on ice, and washing for 3 times with ice PBS; PE-anti human FC antibody was incubated, diluted at 1. PBS 200 μ l/well resuspend cells, read mean fluorescence by FACS instrument, analyze results using graphpad prism software.

The results are shown in figure 1, the anti-CD 3 antibody SP34 and its mutants QP3685, QP3689, QP3690 all retained jurkat binding activity.

Example 3 design of a bispecific antibody format containing anti-TAA (tumor associated antigen) and anti-CD 3

The CD3 double antibody plays a therapeutic role, and mainly binds to a Tumor Associated Antigen (TAA) expressed on a tumor cell and a CD3 target spot on a T cell simultaneously, so that the two cells are crosslinked to form an immune synapse, and the T cell is activated, thereby killing the tumor cell. Multivalent TAA antibodies may improve selectivity for a target, especially if the target is not sufficiently specific. The main direction for improving the safety of the CD3 double antibody is to improve the selectivity of TAA, and meanwhile, the proper CD3 antibody affinity is selected, so that the drug effect can be kept, and the toxic and side effects can be reduced, thereby improving the selectivity of tumors and reducing targeted healthy cells.

The design of the anti-TAA (tumor associated antigen) and anti-CD 3 bispecific antibody molecule format provided by the invention is shown in figure 2.

The anti-TAA and anti-CD 3 bispecific antibody molecules shown in figure 2 comprise: a first single body (left part in fig. 2) and a second single body (right part in fig. 2). The first monomer comprises 2 chains, respectively chain 1: it has Fd fragments (including VH and CH 1); chain 2: it has a light chain fragment (including VL and CL), a first Fc chain. The second monomer also includes 2 strands, respectively strand 1: it has Fd fragments (including VH and CH 1); chain 2: it has a light chain fragment (including VL and CL), an anti-CD 3 Antibody fragment (CD 3 Antibody in figure 2, in scFv form), and a second Fc chain. In the first or second monomer, the light chain fragment and Fd fragment pair to form an anti-TAA Fab domain (for the TAA Antibody function of FIG. 2), respectively. Unlike conventional antibodies, a light chain fragment of a first monomer is fused to a first Fc chain; simultaneously, the N-terminal of the anti-CD 3 antibody fragment is fused to the light chain fragment of the second monomer, and the C-terminal of the anti-CD 3 antibody fragment is fused to the second Fc chain. The first Fc chain and said second Fc chain polymerize to form an Fc domain. The Fc domain may be selected from Human IgG1Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, or variants thereof.

Molecular cloning, transient expression and protein purification of bispecific antibody:

1. molecular cloning:

as shown in fig. 2, TAA selects CLDN18.2 and designs anti-CLDN 18.2/CD3 bispecific antibody, wherein the sequence of CLDN18.2 antibody can be found in the sequence of antibody QP14611463 in patent CN202010344676.8, and the anti-CD 3 antibody uses the sequences of SP34 and its variants QP3685, QP3689 and QP3690 for light and heavy chains of antibody, respectively. anti-CD 3 antibody sequence selection for QP374537493746 the anti-CD 3 antibody sequence selection for SP34, QP090837493746, QP090937493746, QP091037493746 is selected from QP3685, QP3689, QP3690. The protein expression and sequence numbers are shown in the following table.

Table 4: cloning design of bispecific antibodies

Also for comparison, 2 further forms of CLDN18.2/CD3 dual antibody molecules QP372337241461 and QP374437481461 were constructed and CLDN18.2/CD3 dual antibody molecule AMG910 from anshi according to patent US20200055932A1 was constructed, the AMG910 protein being numbered QP3693. The protein expression and sequence numbers are shown in the following table.

Table 5: molecular cloning of bispecific antibodies

The molecular design of QP372337241461 is shown in FIG. 3. The molecular design of QP374437481461 is shown in FIG. 4.

2. The cloning construction method comprises the following steps:

clones were designed as shown in tables 4 and 5, and full-length expression vectors were constructed. Designing a primer: using online software DNAworks (v3.2.4) (V)http://helixweb.nih.gov/dnaworks/) Designing multiple primers to synthesize the gene segment containing the recombination. Segment splicing: the gene fragment required for recombination was obtained by PCR amplification using the primers designed above according to the instruction manual for Primer STAR GXL DNA polymerase from TaKaRa. The first step of PCR: PCR reaction 50. Mu.L included 10. Mu.L PrimerSTAR GXL Buffer (5 ×); 4 μ L dNTP mix (2.5 mmol. Multidot.L-1); primers were 1. Mu.L each as shown above; 1 μ L PrimeSTAR GXL DNA Polymerase. PCR conditions of 98 ℃ for 2min,98 ℃ for 20s,55 ℃ for 15s,68 ℃ for 30s and 30 cycles; 5min at 68 ℃. And a second step of PCR: and (3) performing PCR amplification by using the PCR product of the first step as a template and using the first primer and the last primer under the same conditions as the first step. And constructing and amplifying target fragments by PCR. Constructing an expression vector pQD and carrying out enzyme digestion: the expression vector pQD (signal peptide) is designed and constructed by using the characteristics of some special restriction endonucleases, such as BsmBI, of which the recognition sequence is different from the enzyme cutting site, the BsmBI enzyme-cuts the vector, and the gel is cut and recovered for later use. Recombinant construction of expression vectors: the recombinant target gene fragment and BsmBI enzyme digestion recovery expression vector pQD (signal peptide fragment) are respectively added into DH5 alpha competent cells according to the proportion of 3.

3. Protein expression:

cell density was adjusted to 1X 10 by 293E cells ⁶ And (4) the concentration is/ml. Plasmids and PEI as a transfection reagent were prepared, the amount of plasmid to be transfected was 100ug/100ml cells, and the mass ratio of PEI to plasmid was 2. And mixing the plasmid and PEI evenly, and standing for 15min. The plasmid and PEI mixture was slowly added to 293E cells, which were incubated in a shaker at 37 ℃ with 8% CO2, 120rpm. On days five to six of transfection, horizontal centrifuge at 4700rpm was removedCell supernatants were collected and purified at heart 20 min.

4. Protein purification:

protein affinity chromatography: the cell culture fluid is centrifuged at high speed, and the supernatant is taken out and subjected to affinity chromatography by using a Protein A chromatographic column of GE. Performing chromatography with 1 × PBS (pH7.4) as balance buffer, washing cell supernatant with PBS until ultraviolet ray returns to baseline, eluting with 0.1M glycine (pH3.0) as elution buffer, and adjusting pH to neutral with Tris; protein size exclusion chromatography: the product obtained by ion exchange is concentrated by ultrafiltration and then subjected to size exclusion chromatography, for example separation on GE Superdex200 gel, to remove possible aggregates and other components and obtain the desired product of high purity. The purity of the protein obtained can be analyzed by SDS-PAGE and SEC-HPLC detection. Protein concentration was determined by uv spectrophotometry. The endotoxin is strictly controlled in the whole purification process, and the purified protein endotoxin content is less than 1EU/mg.

The results show that the CLDN18.2/CD3 bispecific antibody QP374537493746, QP090837493746, QP090937493746 and QP091037493746 have high cell expression yield, HPLC-SEC purity of more than 95%, and have producibility.

Example 4: FACS detection of bispecific antibody binding Activity to CLDN18.2

The experimental steps are as follows:

taking a cell strain CHOS-CLDN18.2 stably expressing CLDN18.2 and a seed 1E 5/hole cell CHOS-CLDN18.2 in a U-shaped 96-well plate, washing once with ice PBS, centrifuging at 1200rpm for 3min; after washing, 3% FBS/PBS blocking solution was added at 200. Mu.L/well, and incubated on ice for 1h. After sealing, centrifuging at 1200rpm for 3min, throwing off the supernatant, incubating samples with different concentrations, and incubating on ice for 2h and washing with ice PBS for 3 times; PE-anti human FC antibody was incubated, diluted at 1. PBS 200 μ l/well resuspend cells, read mean fluorescence by FACS instrument, analyze results using graphpad prism software.

As shown in fig. 5 and 6, the results show that: CLDN18.2/CD3 bispecific antibodies QP374537493746, QP090837493746, QP090937493746 and QP091037493746 bind to CLDN18.2. As shown in fig. 5, the affinity of the anti-CLDN 18.2 forms of the invention, di-anti-QP 374537493746, QP090837493746, QP090937493746 and QP091037493746, for binding TAA target CLDN18.2, was comparable to anti-CLDN 18.2 antibody IgG form QP14611463, significantly stronger than the control antibody AMG910 (1 form) QP3693, consistent with design expectations.

Example 5: FACS detection of the binding Activity of bispecific antibodies to CD3

Human lymphocytic leukemia cells (Jurkat cells) express native CD3 and binding of anti-CD 3 antibodies and mutants thereof to Jurkat cells is detected by FACS. Seeding 1E 5/well Jurkat cells in a U-shaped 96-well plate, washing once with ice PBS (phosphate buffer solution), centrifuging at 1200rpm for 3min; after washing, 3% FBS/PBS blocking solution was added at 200. Mu.L/well, and incubated on ice for 1h. After sealing, centrifuging at 1200rpm for 3min, throwing off the supernatant, incubating samples with different concentrations, incubating for 2h on ice, and washing for 3 times with ice PBS; PE-anti human FC antibody was incubated, diluted at 1. PBS 200 μ l/well resuspend cells, read mean fluorescence by FACS instrument, analyze results using graphpad prism software.

The results are shown in fig. 7 and fig. 8, and show that CLDN18.2/CD3 bispecific antibodies QP374537493746, QP090837493746, QP090937493746 and QP091037493746 all bind to CD3. As shown in fig. 7, the dual anti-CD antibodies of the invention of the form 2, QP374537493746, QP090837493746, QP090937493746 and QP091037493746, are classified into 2 classes in terms of their binding affinity to CD3, wherein QP374537493746, QP090837493746, and QP090937493746 have a weaker affinity to CD3, and QP 0909374746 and QP091037493746 are stronger, but these 2 classes of molecules have a significantly weaker affinity to CD3 than the control antibody AMG910 (form 1) QP3693, which is in line with our expectation that we would like to reduce the affinity of the CD3 antibody to improve the CD3 dual anti-safety.

Example 6: CLDN18.2/CD3 bispecific antibody mediated T cell killing of human gastric cancer cells

The experimental method comprises the following steps: the target cells were selected as human gastric cancer cell line NUGC4-CLDN18.2 stably expressing CLDN18.2. The effector cells are human PBMCs. According to the proportion of E to T of 10:1, adding antibodies at different concentrations, 37 ℃,5% CO ₂ Culturing for 48h, using CytoTox

Non-Radioactive cytotoxin Assay (Promega, G1780-1000 assays) detects LDH in cell culture supernatant and quantitates Cytotoxicity%. Maximum lysis rate of target cells (100%) 1% Triton X-100 treatment of target cells lyses the cells releasing all LDH. And setting control holes of target cell spontaneous, effector cell spontaneous, target cell + effector cell spontaneous and the like. According to the formula% cytoxicity = [ (Experimental-Effector cyclone-Target cyclone)/(Target Maximum-Target cyclone)]X 100 analysis of data.

As shown in FIGS. 9 and 10, QP374537493746, QP090837493746, QP090937493746 and QP091037493746 mediated killing of PBMC to human gastric cancer cells NUGC4-CLDN18.2 cells. As shown in FIG. 9, the cell killing activity of human gastric cancer cells NUGC4-CLDN18.2 mediated by the forms of the invention 2, namely the double antibodies QP374537493746, QP090837493746, QP 0909374746 and QP091037493746, is stronger than that of a control molecule QP3693 (AMG 910). Although all of the selected CD3 antibodies of the present invention bind CD3 with weaker affinity than the CD3 antibody of AMG910 molecule (see fig. 7), they do not affect the killing ability on target cells.

Example 7: CLDN18.2/CD3 bispecific antibody mediated T cell killing of human lung cancer cells

The experimental method comprises the following steps: the target cells were selected as human lung cancer cell line HCC827-CLDN18.2 stably expressing CLDN18.2. The effector cells are human PBMCs. According to the proportion of E to T of 10:1, adding antibody of different concentrations, culturing at 37 deg.C and 5% in CO2 for 48h, and culturing with CytoTox

Non-Radioactive cytotoxin Assay (Promega, G1780-1000 assays) detects LDH in cell culture supernatant and quantitates Cytotoxicity%. Maximum lysis rate of target cells (100%) 1% Triton X-100 treatment of target cells lyses the cells releasing all LDH. Control wells for target cell spontaneous, effector cell spontaneous, target cell + effector cell spontaneous, and the like are set. According to the formula% cytoxicity = [ (Experimental-effective-Effector species-Target species)/(Target Maximum-Target species)]X 100 analysis of data.

The results are shown in FIG. 11, CD3 double-anti molecules QP374537493746, QP090837493746, QP090937493746 and QP091037493746 can mediate PBMC killing human lung cancer cells HCC827-CLDN18.2 cells, and the activities of the invention in the form of FIG. 2 double-anti molecules QP 374537493493746, QP 837493746, QP090937493746 and QP091037493746 mediating PBMC killing human lung cancer cells HCC827-CLDN18.2 cells are stronger than the activity of the control molecule QP3693 (AMG 910). The results of NUGC4-CLDN18.2 were reproduced on HCC827-CLDN18.2 cells (see FIG. 9).

Example 8: CD3 humanized mouse model animal drug effect

The inhibitory effect of the anti-CLDN 18.2/CD3 dual antibody molecule on tumor growth was evaluated in the CD3 humanized mouse (CD 3EDG HuGEMM) model. Collecting the colorectal cancer cells MC 38-hLDN 18.2 cells in the exponential growth phase, and resuspending the cells to a proper concentration by PBS for inoculation. Each experimental mouse was inoculated subcutaneously on the right back with 5X 10 ⁶ MC 38-hLDN18.2 cells, periodically observing the growth of the tumor, and waiting until the tumor grows to an average volume of about 100mm ³ The administration was randomized in groups based on tumor size and mouse weight. The dosing regimen was as follows:

table 6: dosing regimen for evaluating the inhibitory effect of anti-CLDN 18.2/CD3 dual anti-molecule on tumor growth

The results, which are shown in table 7 and the tumor inhibition curves are shown in fig. 12, show that the tumor inhibition ratio (TGI) of QP374537493746 in the low dose group of 0.6mpk reached 89.29% at day 21 after administration, which is superior or equivalent to the tumor inhibition ratio (TGI = 86.5%) of control antibody AMG910 in the 0.7mpk group (the molar concentration of AMG910 in the 0.7mpk group is equivalent to that of qp3745374937461.2mpk), i.e. half the dose of QP374537493746 can achieve the same tumor inhibition effect as the control antibody AMG910. Fig. 13 shows the body weight change of each group of mice after administration, and the results show that the body weight of the mice in the administration group has almost no change, the growth is good, and the safety of the drug is good.

Table 7: CLDN18.2/CD3 dual antibody molecule inhibits tumor growth in an in vivo animal model of subcutaneous transplantation of colorectal cancer cells MC38-hCLDN18.2 in CD3EGD HuGEMM mice

*Note:％T/C＝(delta T/delta C)×100,％TGI＝100-％T/C.

Example 9: humanized NOG mouse model animal drug effect

Purpose of the experiment: the test subjects were evaluated for in vivo efficacy in a PBMC humanized NOG mouse X-CLDN18.2/MIA PaCa-2 subcutaneous graft tumor model. And (3) cell culture: X-CLDN18.2/MIA PaCa-2 cells are cultured in vitro in a medium supplemented with 10% fetal bovine serum, 37 ℃,5% ₂ . Routinely passaged 2 times a week. When the cells are maintained in exponential growth phase and the cell survival rate is greater than 95%, the cells are harvested, counted, the percentage of viable cells is counted and inoculated. The animals are NOG mice, female, 6-8 weeks old and 18-20 g in weight. The total amount of the experiment is 36 (24plus 50%). Animals were provided by qualified suppliers. Tumor inoculation: mixed inoculation 5 x 10 in the right dorsum cervices of each mouse ⁶ X-CLDN18.2/MIA PaCa-2 and 2X 10 ⁶ And (4) PBMC cells. And (3) marking the experimental animal with an ear tag number while inoculating, and taking the marked animal as a unique confirmation mark of a subsequent experiment. Waiting for tumor growth until the average tumor volume reaches about 60-100mm ³ Random group administration was started. The experimental groups and dosing regimen are shown in table 8.

Table 8: dosing regimen for evaluating the inhibitory effect of an anti-CLDN 18.2/CD3 dual antibody molecule on tumor growth

Following the protocol shown in the table above, 5 doses were administered 2 times per week with tumor inhibition rates as shown in table 9 and tumor inhibition curves as shown in fig. 14, showing that the results at day 15 after administration showed that QP374537493746 reached 92.3% of tumor inhibition rate (TGI) at 4mpk in the low dose group and =99.8% of tumor inhibition rate (TGI) at 8mpk in the high dose group, both significantly better than the control antibody AMG910 at 5mpk group (molar concentration of AMG910 5mpk group is equivalent to that of QP 3749337493746 at 8mpk in the high dose group) (TGI = 69.3%). Fig. 15 shows the body weight changes of mice in each group after administration, and the results show that the body weight of mice in the administration group is almost unchanged, the growth is good, and the safety of the drug is good.

Table 9: CLDN18.2/CD3 dual antibody molecule inhibits tumor growth in a PBMC humanized NOG mouse X-CLDN18.2/MIA PaCa-2 subcutaneous graft tumor model

*Note:％T/C＝(delta T/delta C)×100,％TGI＝100-％T/C.

II, secondly: anti-TAA (tumor associated antigen), anti-CD 3 antibody and multifunctional fusion protein form design containing cytokine IL15/IL15Ra

Although CD3 dual antibodies show great potential in hematologic tumors, CD3 dual antibodies still face several challenges in the treatment of solid tumors. The CD3 double-antibody treatment is related to T cell infiltration and immunosuppression of solid tumor tissues, and the CD3 double-antibody treatment is easily resisted under the condition that the infiltrated T cells are few. IL15 is a soluble cytokine that activates T cells and NK cells and mediates proliferation and survival of these cells. The invention designs a TAA/CD3/IL15 multifunctional fusion protein, so that a cytokine IL15 component in the fusion protein can stimulate immune cells including T cells to proliferate and change the immune microenvironment of tumors.

Example 10: anti-TAA (tumor associated antigen), anti-CD 3 and multifunctional fusion protein form design containing IL15/IL15Ra

The invention provides various anti-TAA (tumor associated antigen), anti-CD 3 and multifunctional fusion proteins containing IL15/IL15 Ra.

1. A first type of multifunctional fusion protein can be seen in FIGS. 16-19, where the fusion protein molecule includes a first monomer (left portion of the figure) and a second monomer (right portion of the figure). The first monomer comprises 2 chains, respectively chain 1: it has Fd fragments (including VH and CH 1); chain 2: it has a light chain fragment (including VL and CL), a cytokine domain (including IL15 and IL15 Ra), a first Fc chain. The second monomer comprises 2 chains, respectively chain 1: it has Fd fragments (including VH and CH 1); chain 2: it has a light chain fragment (including VL and CL), an anti-CD 3 Antibody fragment (CD 3 Antibody in the figure, which is in scFv form), and a second Fc chain. In the first monomer or the second monomer, the light chain fragment and Fd fragment are paired to form an Anti-TAA Fab domain (for the Anti-TAA Antibody function in the figure), respectively. The N-terminal of the cytokine functional region is fused with the light chain fragment of the first monomer, and the C-terminal is fused with the first Fc chain; the N-terminus of the anti-CD 3 antibody fragment is fused to the light chain fragment of the second monomer and the C-terminus is fused to the second Fc chain. The first Fc chain and the second Fc chain polymerize to form an Fc domain.

In fig. 16 to 19, the anti-CD 3 antibody fragment in scFv format may include: a heavy chain variable region (VH), a light chain variable region (VL), and a linker linking the heavy chain variable region and the light chain variable region. The fusion sequence of the amino acids of the anti-CD 3 antibody fragment from the N-terminus to the C-terminus of the peptide chain may be "VH (CD 3) to VL (CD 3)", or "VL (CD 3) to VH (CD 3)"; "-" indicates a connection fragment. The fusion sequence of the amino acid fragments of the functional domains of cytokines may be "IL-15 to IL-15Ra", or "IL-15Ra to IL-15".

The 4 polypeptide chains of the fusion protein shown in FIG. 16 can be described as follows: (1) a first polypeptide chain: VH-CH1; (2) a second polypeptide chain: VL-CL-IL15Ra-IL15-a first Fc chain; (3) a third polypeptide chain: VL-CL-VH(CD3)-VL(CD3)-a second Fc chain; (4) a fourth polypeptide chain: VH-CH1."-" indicates ligation by peptide bond or linker fragment.

The 4 polypeptide chains of the fusion protein shown in FIG. 17 are depicted as follows: (1) a first polypeptide chain: VH-CH1; (2) a second polypeptide chain: VL-CL-IL15-IL15Ra-a first Fc chain; (3) a third polypeptide chain: VL-CL-VH(CD3)-VL(CD3)-a second Fc chain; (4) a fourth polypeptide chain: VH-CH1.

The 4 polypeptide chains of the fusion protein shown in FIG. 18 are depicted as follows: (1) a first polypeptide chain: VH-CH1; (2) a second polypeptide chain: VL-CL-IL15Ra-IL15-a first Fc chain; (3) a third polypeptide chain: VL-CL-VL(CD3)VH(CD3)-a second Fc chain; (4) a fourth polypeptide chain: VH-CH1.

The 4 polypeptide chains of the fusion protein shown in FIG. 19 are depicted below: (1) a first polypeptide chain: VH-CH1; (2) a second polypeptide chain: VL-CL-IL15-IL15Ra-a first Fc chain; (3) a third polypeptide chain: VL-CL-VL(CD3)VH(CD3)-a second Fc chain; (4) a fourth polypeptide chain: VH-CH1.

2. A second type of multifunctional fusion protein can be seen in FIGS. 20-27, the protein molecules comprising: a first cell (left part in the figure) and a second cell (right part in the figure). The first monomer includes: an anti-TAA antibody fragment (in scFv format, comprising VH and VL domains), a cytokine functional region (comprising IL15 and IL15 Ra), a first Fc chain; the second monomer comprises: an anti-TAA Antibody fragment (in scFv format, comprising VH and VL domains), an anti-CD 3 Antibody fragment (in the figure, CD3 Antibody, in scFv format), and a second Fc chain. The N-terminal of the cytokine functional region is fused with the anti-TAA antibody fragment of the first monomer, and the C-terminal is fused with the first Fc chain; the N-terminus of the anti-CD 3 antibody fragment is fused to the anti-TAA antibody fragment of the second monomer and the C-terminus is fused to the second Fc chain. The first Fc chain and the second Fc chain polymerize to form an Fc domain.

Similar to the first type of multifunctional fusion protein, the anti-CD 3 antibody fragment in scFv format can include: a heavy chain variable region (VH), a light chain variable region (VL), and a linker linking the heavy chain variable region and the light chain variable region. The fusion sequence of the amino acids of the anti-CD 3 antibody fragment from the N-terminus to the C-terminus of the peptide chain may be "VH (CD 3) to VL (CD 3)", or "VL (CD 3) to VH (CD 3)"; "-" indicates the ligated fragment. The fusion sequence of the amino acid fragments of the cytokine domain may be "IL-15 to IL-15Ra", or "IL-15 to IL-15". In addition, anti-TAA antibody fragments in scFv format include: a heavy chain variable region (VH), a light chain variable region (VL), and a linker linking the heavy chain variable region and the light chain variable region; the amino acid fusion sequence of the anti-TAA antibody fragment may be "VH (TAA) -VL (TAA)" or "VL (TAA) -VH (TAA)" from the N-terminus to the C-terminus of the peptide chain; "-" indicates the ligated fragment.

The antibody molecules shown in FIGS. 20-27 each employ the same amino acid sequence, differing in the order of fusion or configuration. Using FIG. 20 as an example, the following are depicted for the 2 polypeptide chains of the fusion protein: (1) a first polypeptide chain: VL (TAA) -VH (TAA) -IL15 Ra-first Fc chain; (2) a second polypeptide chain: VL (TAA) -VH (CD 3) -VL (CD 3) -second Fc chain.

3. The present invention also provides a tumor-targeted T cell/NK cell activated TAA/IL15 bi-functional fusion protein, the structural form of which can be seen in fig. 28, which is distinguished by the replacement of the anti-CD 3 antibody fragment with a cytokine domain (including IL15 and IL15 Ra) as compared to the second type of multifunctional fusion protein. In fig. 28, the protein molecule has a symmetrical structural form.

Example 11: molecular cloning, transient expression and protein purification of CLDN18.2/CD3/IL15 multifunctional fusion protein

1. Molecular cloning:

according to example 10, CLDN18.2/CD3/IL15 or CLDN18.2/IL15 multifunctional fusion proteins are designed, wherein the CLDN18.2 antibody can refer to the QP14611463 sequence of the antibody in the patent document CN202010344676.8, and the SP34 light and heavy chain sequence is used as the anti-CD 3 antibody, and the protein expression and the sequence number are shown in the following table.

Table 10: cloning design of multifunctional fusion proteins

IL15/IL15Ra-FC from Henry pharmaceutical Co was constructed as a control, and the IL15/IL15Ra-FC protein was numbered QP33123313.

Clones were designed as shown in table 10 and full-length expression vectors were constructed. The cloning construction, protein expression and protein purification methods can be found in example 3.

Example 12: FACS detection of CLDN18.2/CD3/IL15 multifunctional fusion protein binding activity to CLDN18.2

The experimental steps are as follows: taking a cell strain CHOS-CLDN18.2 stably expressing CLDN18.2 and a seed 1E 5/hole cell CHOS-CLDN18.2 in a U-shaped 96-well plate, washing once with ice PBS, centrifuging at 1200rpm for 3min; after washing, 200. Mu.L/well of 3% FBS/PBS blocking solution was added, and incubated on ice for 1h. After sealing, centrifuging at 1200rpm for 3min, throwing off the supernatant, incubating samples with different concentrations, incubating for 2h on ice, and washing for 3 times with ice PBS; the PE-anti human FC antibody was incubated, diluted at a rate of 1. PBS 200 ul/well resuspend cells, FACS instrument read mean fluorescence, analyze results using graphpad prism software. The results are shown in fig. 29, fig. 30, and show that both CLDN18.2/CD3/IL15 and CLDN18.2/IL15 multifunctional fusion proteins bind to TAA target CLDN18.2 and have stronger affinity than AMG910 analog (form 1) QP3693, as expected.

Example 13: FACS detection of binding Activity of CLDN18.2/CD3/IL15 multifunctional fusion protein to CD3

Lymphocytic leukemia cells (Jurkat cells) express native CD3 and binding of anti-CD 3 antibodies and mutants thereof to Jurkat cells is detected by FACS. Seeding 1E 5/well Jurkat cells in a U-shaped 96-well plate, washing once with ice PBS (phosphate buffer solution), centrifuging at 1200rpm for 3min; after washing, 200. Mu.L/well of 3% FBS/PBS blocking solution was added, and incubated on ice for 1h. After sealing, centrifuging at 1200rpm for 3min, throwing off the supernatant, incubating samples with different concentrations, incubating for 2h on ice, and washing for 3 times with ice PBS; PE-anti human FC antibody was incubated, diluted at 1. PBS 200 ul/well resuspended cells, FACS machine read mean fluorescence values, and the results were analyzed using graphpad prism software. The results are shown in fig. 31, fig. 32, and show that the CLDN18.2/CD3/IL15 multifunctional fusion proteins all bind CD3 and have weaker affinity than the control molecule AMG910 (QP 3693), which is consistent with the expectation that the inventors expect a reduction in the affinity of the CD3 antibody to improve the safety of the CD3 dual antibody.

Example 14: mo7e cell proliferation experiment evaluation of IL15 activity of multifunctional fusion protein

Mo7e (human cytomegalo leukemia cell line) cells express IL-15R beta gamma, and are cytokine growth-dependent cells. Studies have shown that resting NK and naive T cells express a medium affinity IL-15R β γ phenotype, and that the results of experiments on Mo7e (IL-15R β γ) cell proliferation by the cytokine IL15/IL15Ra are consistent with those on unstimulated PBMC (Mol Cancer Ther;11 (6) June 2012). The invention uses Mo7e cell proliferation experiment to evaluate the activity of multifunctional fusion protein IL15, and the method and the result are as follows:

experimental reagent: mo7e cells (human giant cell leukemia cell strains) purchased from the center of cell resources of the institute of basic medicine of Chinese academy of medical sciences, cell proliferation and toxicity detection kit (CCK-8) purchased from Melam bioscience, cat # MA0218; recombinant human GM-CSF, available from perprotech, cat # 300-03; human IgG, purchased from Sigma, cat # I4506; other antibodies are derived from internal preparations.

The experimental method comprises the following steps: mo7e cells were 5% CO at 37 ℃ using RPMI1640 culture containing 10% FBS,2mM L-glutamine and 8ng/ml GM-CSF ₂ Culturing in an incubator; mo7e cells are collected, centrifuged at 800rpm for 5 minutes, the supernatant is poured off, and the cells are washed 2 times by RPMI1640 medium without GM-CSF; resuspend cells in RPMI1640 Medium without GM-CSF and enumerate the cells by 2X 10 ⁴ 80 μ l per well in 96-well plates, 37 ℃ 5% CO ₂ Culturing for 1 hour in an incubator; diluting each of the medicinal culture media to be tested in 4-fold gradient, mixing 20 μ l each well with the cell suspension, and reacting at 37 deg.C with 5% CO ₂ Culturing in an incubator for 3 days; adding 10. Mu.l of CCK-8 reagent per well to a 96-well plate to be tested, 37 ℃ 5% ₂ Incubating for 4 hours in an incubator; the 96-well plate was taken out and the absorbance at a wavelength of 450nm was measured in a microplate reader.

The results of the experiments are shown in fig. 33, fig. 34 and fig. 35, and show that the CLDN18.2/CD3/IL15 and CLDN18.2/IL15 multifunctional fusion proteins with different designs all have IL15 activity, and the activity is strong or weak according to different molecular designs. Different constructs produce molecules with different IL15 activity, which provides the opportunity to select the best therapeutic window.

Example 15: CLDN18.2/CD3/IL15 multifunctional fusion protein mediated T cell killing human gastric cancer cell

Non-Radioactive cytotoxin Assay (Promega, G1780-1000 assays) detects LDH in cell culture supernatant and quantitates Cytotoxicity%. Maximum lysis rate of target cells (100%) 1% Triton X-100 treatment of target cells lyses the cells releasing all LDH. Control wells for target cell spontaneous, effector cell spontaneous, target cell + effector cell spontaneous, and the like are set. According to the formula% cytoxicity = [ (Experimental-Effector cyclone-Target cyclone)/(Target Maximum-Target cyclone)]X 100 analysis of data. As shown in FIGS. 36 and 37, QP374508993746, QP090109003746, QP09023688, QP36670903, QP09040905, QP09060905, QP09040907 and QP09060907 mediate the killing of human gastric cancer NUGC4-CLDN18.2 cells by PBMC. Wherein QP374508993746 and QP090109003746 mediate the activity of PBMC to kill human lung cancer cell NUGC4-CLDN18.2 cells to be obviously better than the control molecule AMG910. The cell killing activity of human lung cancer cells NUGC4-CLDN18.2 mediated by PBMCs of other molecules is equivalent to that of the control molecule AMG910.

Example 16: human lung cancer cell killing by T cell mediated by CLDN18.2/CD3/IL15 multifunctional fusion protein

The experimental method comprises the following steps: the target cells were selected as human lung cancer cell line HCC827-CLDN18.2 stably expressing CLDN18.2. The effector cells are human PBMCs. According to the proportion of E to T of 10:1, adding antibodies at different concentrations, 37 ℃,5% ₂ Culturing for 48h, using CytoTox

Non-Radioactive cytotoxin Assay (Promega, G1780-1000 assays) detects LDH in cell culture supernatant and quantitates Cytotoxicity%. The maximum lysis rate (100%) of the target cells was 1% treatment of the target cells with Triton X-100 lysed the cells to release all LDH. Setting target cellsSpontaneous, effector cell spontaneous, target cell + effector cell spontaneous, etc. control wells. According to the formula% cytoxicity = [ (Experimental-Effector cyclone-Target cyclone)/(Target Maximum-Target cyclone)]X 100 analysis of data. The results are shown in FIG. 38, QP090109003746, QP36670903, QP09040905, QP09060905 and QP09040907 can mediate the killing activity of PBMC to human lung cancer cells HCC827-CLDN18.2 cells. Wherein, QP090109003746 mediated PBMC kills human lung cancer cells HCC827-CLDN18.2 cell activity is obviously better than that of a control molecule AMG910. The activities of other molecules mediated PBMC killing human lung cancer cell HCC827-CLDN18.2 cells are comparable to the control molecule AMG910.

In addition, in some other examples, the anti-CD 3 antibody fragment was selected from the light and heavy chain sequences of antibodies of SP34 variants QP3685, QP3689, QP3690, respectively, and the protein was cloned and expressed and purified as shown in table 10 in example 11. The results show that the molecules constructed by the light and heavy chain sequences of QP3685, QP3689 and QP3690 antibodies also have the CLDN18.2 binding activity and the IL15 activity, and the anti-CD 3 molecules also have the CD3 binding activity and have the effect of mediating T cells to kill human gastric cancer cells or lung cancer cells.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be limited only by the attached claims.

Sequence listing

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Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val

130 135 140

Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu

145 150 155 160

Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

165 170 175

Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly

180 185 190

Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu

195 200 205

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp

210 215 220

Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

225 230 235 240

Gly Gly Gly Thr Lys Leu Thr Val Leu Glu Pro Lys Ser Ser Asp Lys

245 250 255

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

260 265 270

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

275 280 285

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

290 295 300

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

305 310 315 320

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

325 330 335

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

340 345 350

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

355 360 365

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

370 375 380

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

385 390 395 400

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

405 410 415

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

420 425 430

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

435 440 445

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

450 455 460

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470 475 480

Lys

<210> 5

<211> 740

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser

245 250 255

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala

260 265 270

Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln

275 280 285

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr

290 295 300

Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr

305 310 315 320

Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn

325 330 335

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn

340 345 350

Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr

355 360 365

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly Gly Gly Ser Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr

385 390 395 400

Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly

405 410 415

Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly

420 425 430

Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly

435 440 445

Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu

450 455 460

Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala

465 470 475 480

Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr

485 490 495

Val Leu Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro

500 505 510

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

515 520 525

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

530 535 540

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

545 550 555 560

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

565 570 575

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

580 585 590

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

595 600 605

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

610 615 620

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

625 630 635 640

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

645 650 655

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

660 665 670

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

675 680 685

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

690 695 700

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

705 710 715 720

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser Ser His His

725 730 735

His His His His

740

<210> 6

<211> 478

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu

225 230 235 240

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

370 375 380

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Leu Gly Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475

<210> 7

<211> 242

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys

<210> 8

<211> 740

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser

245 250 255

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala

260 265 270

Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln

275 280 285

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr

290 295 300

Asn Asn Tyr Glu Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr

305 310 315 320

Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn

325 330 335

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn

340 345 350

Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr

355 360 365

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly Gly Gly Ser Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr

385 390 395 400

Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly

405 410 415

Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly

420 425 430

Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Leu Arg Ala Pro Gly

435 440 445

Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu

450 455 460

Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala

465 470 475 480

Leu Trp Arg Ser Gly Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr

485 490 495

Val Leu Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro

500 505 510

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

515 520 525

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

530 535 540

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

545 550 555 560

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

565 570 575

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

580 585 590

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

595 600 605

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

610 615 620

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

625 630 635 640

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

645 650 655

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

660 665 670

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

675 680 685

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

690 695 700

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

705 710 715 720

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser Ser His His

725 730 735

His His His His

740

<210> 9

<211> 740

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser

245 250 255

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala

260 265 270

Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln

275 280 285

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr

290 295 300

Asn Asn Tyr Glu Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr

305 310 315 320

Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn

325 330 335

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Ser Asn

340 345 350

Phe Gly Asn Gly Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr

355 360 365

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly Gly Gly Ser Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr

385 390 395 400

Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly

405 410 415

Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly

420 425 430

Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Leu Arg Ala Pro Gly

435 440 445

Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu

450 455 460

Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala

465 470 475 480

Leu Trp Asn Lys Gly Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr

485 490 495

Val Leu Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro

500 505 510

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

515 520 525

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

530 535 540

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

545 550 555 560

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

565 570 575

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

580 585 590

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

595 600 605

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

610 615 620

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

625 630 635 640

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

645 650 655

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

660 665 670

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

675 680 685

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

690 695 700

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

705 710 715 720

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser Ser His His

725 730 735

His His His His

740

<210> 10

<211> 740

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser

245 250 255

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala

260 265 270

Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln

275 280 285

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr

290 295 300

Asn Asn Tyr Glu Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr

305 310 315 320

Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn

325 330 335

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Ser Asn

340 345 350

Phe Gly Asn Gly Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr

355 360 365

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly Gly Gly Ser Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr

385 390 395 400

Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly

405 410 415

Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly

420 425 430

Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Trp Leu Arg Ala Pro Gly

435 440 445

Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu

450 455 460

Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala

465 470 475 480

Leu Trp Asn Ser Gly Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr

485 490 495

Val Leu Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro

500 505 510

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

515 520 525

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

530 535 540

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

545 550 555 560

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

565 570 575

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

580 585 590

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

595 600 605

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

610 615 620

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

625 630 635 640

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

645 650 655

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

660 665 670

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

675 680 685

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

690 695 700

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

705 710 715 720

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser Ser His His

725 730 735

His His His His

740

<210> 11

<211> 636

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

245 250 255

Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

260 265 270

Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr

275 280 285

Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

290 295 300

Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala

305 310 315 320

Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn

325 330 335

Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val

340 345 350

Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp

355 360 365

Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys

385 390 395 400

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

405 410 415

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

420 425 430

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

435 440 445

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

450 455 460

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

465 470 475 480

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

485 490 495

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

500 505 510

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

515 520 525

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

530 535 540

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

545 550 555 560

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

565 570 575

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp

580 585 590

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

595 600 605

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

610 615 620

Gly Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp Lys

625 630 635

<210> 12

<211> 618

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

245 250 255

Ser Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly

260 265 270

Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

275 280 285

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg

290 295 300

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg

305 310 315 320

Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly

325 330 335

Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser

340 345 350

Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys

370 375 380

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

385 390 395 400

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

405 410 415

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

420 425 430

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

435 440 445

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

450 455 460

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

465 470 475 480

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

485 490 495

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

500 505 510

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

515 520 525

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

530 535 540

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

545 550 555 560

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

565 570 575

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

580 585 590

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

595 600 605

Gly Gly Ser Ser His His His His His His

610 615

<210> 13

<211> 242

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser

20 25 30

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

35 40 45

Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr

50 55 60

Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser

65 70 75 80

Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala

100 105 110

Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln

115 120 125

Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe

130 135 140

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

145 150 155 160

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

165 170 175

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

180 185 190

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

195 200 205

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

210 215 220

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

225 230 235 240

Glu Cys

<210> 14

<211> 743

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

245 250 255

Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu

260 265 270

Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp

275 280 285

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg

290 295 300

Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp

305 310 315 320

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln

325 330 335

Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg

340 345 350

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly

355 360 365

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val Thr Gln Glu Pro

385 390 395 400

Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser

405 410 415

Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln

420 425 430

Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg

435 440 445

Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys

450 455 460

Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr

465 470 475 480

Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr

485 490 495

Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro

500 505 510

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

515 520 525

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

530 535 540

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

545 550 555 560

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

565 570 575

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

580 585 590

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

595 600 605

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

610 615 620

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

625 630 635 640

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

645 650 655

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

660 665 670

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

675 680 685

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

690 695 700

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

705 710 715 720

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser

725 730 735

Ser His His His His His His

740

<210> 15

<211> 481

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

245 250 255

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

340 345 350

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

370 375 380

Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

420 425 430

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Leu Gly Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp

465 470 475 480

Lys

<210> 16

<211> 990

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Ile Thr Val Ala Gly Thr Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

130 135 140

Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Gly Val Asn Asn Trp Leu Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Thr Ala Ser

180 185 190

Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile Thr Phe Gly Cys

225 230 235 240

Gly Thr Arg Leu Glu Ile Lys Ser Gly Gly Gly Gly Ser Glu Val Gln

245 250 255

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys

260 265 270

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr Ala Met Asn

275 280 285

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile

290 295 300

Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys

305 310 315 320

Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu

325 330 335

Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val

340 345 350

Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr Trp

355 360 365

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

370 375 380

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val Thr Gln Glu

385 390 395 400

Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly

405 410 415

Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn Trp Val Gln

420 425 430

Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Lys Phe

435 440 445

Leu Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly

450 455 460

Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu

465 470 475 480

Tyr Tyr Cys Val Leu Trp Tyr Ser Asn Arg Trp Val Phe Gly Gly Gly

485 490 495

Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Asp Lys Thr His Thr Cys

500 505 510

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

515 520 525

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

530 535 540

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

545 550 555 560

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

565 570 575

Pro Cys Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Cys Val Ser Val Leu

580 585 590

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

595 600 605

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

610 615 620

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

625 630 635 640

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

645 650 655

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

660 665 670

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

675 680 685

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

690 695 700

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

705 710 715 720

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly

725 730 735

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

740 745 750

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr

755 760 765

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

770 775 780

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

785 790 795 800

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

805 810 815

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

820 825 830

Lys Pro Cys Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Cys Val Ser Val

835 840 845

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

850 855 860

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

865 870 875 880

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

885 890 895

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

900 905 910

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

915 920 925

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

930 935 940

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

945 950 955 960

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

965 970 975

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

980 985 990

<210> 17

<211> 692

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met

245 250 255

Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser

260 265 270

Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr

275 280 285

Cys Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His

290 295 300

Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Ser Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn Trp

325 330 335

Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser

340 345 350

Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser

355 360 365

Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile

370 375 380

Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu

385 390 395 400

Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu

405 410 415

Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu

420 425 430

Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

435 440 445

Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

450 455 460

Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

465 470 475 480

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

485 490 495

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

500 505 510

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

515 520 525

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

530 535 540

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

545 550 555 560

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

565 570 575

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

580 585 590

Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

595 600 605

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

610 615 620

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

625 630 635 640

Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

645 650 655

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

660 665 670

Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser Ser Asp Tyr Lys Asp

675 680 685

Asp Asp Asp Lys

690

<210> 18

<211> 697

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Asn Trp Val Asn Val Ile Ser Asp

245 250 255

Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr

260 265 270

Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met

275 280 285

Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Cys Glu Ser Gly Asp

290 295 300

Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn

305 310 315 320

Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys

325 330 335

Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val

340 345 350

His Ile Val Gln Met Phe Ile Asn Thr Ser Ser Gly Gly Gly Ser Gly

355 360 365

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

370 375 380

Gly Ser Gly Gly Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala

385 390 395 400

Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile

405 410 415

Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu

420 425 430

Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser

435 440 445

Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro

450 455 460

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

465 470 475 480

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

485 490 495

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

500 505 510

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

515 520 525

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

530 535 540

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

545 550 555 560

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

565 570 575

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

580 585 590

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala

595 600 605

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

610 615 620

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

625 630 635 640

Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg

645 650 655

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

660 665 670

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser

675 680 685

Ser Asp Tyr Lys Asp Asp Asp Asp Lys

690 695

<210> 19

<211> 740

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val Thr Gln Glu

245 250 255

Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg

260 265 270

Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln

275 280 285

Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys

290 295 300

Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly

305 310 315 320

Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu

325 330 335

Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly

340 345 350

Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

355 360 365

Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

370 375 380

Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe

385 390 395 400

Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys

405 410 415

Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala

420 425 430

Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp

435 440 445

Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu

450 455 460

Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser

465 470 475 480

Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

485 490 495

Ser Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro

500 505 510

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

515 520 525

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

530 535 540

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

545 550 555 560

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

565 570 575

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

580 585 590

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

595 600 605

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

610 615 620

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

625 630 635 640

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

645 650 655

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

660 665 670

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

675 680 685

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

690 695 700

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

705 710 715 720

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser Ser His His

725 730 735

His His His His

740

<210> 20

<211> 731

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

145 150 155 160

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

165 170 175

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

180 185 190

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

195 200 205

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

210 215 220

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile

275 280 285

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

290 295 300

Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr

305 310 315 320

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Cys Glu Ser

325 330 335

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

340 345 350

Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys

355 360 365

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

370 375 380

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Ser Gly Gly Gly

385 390 395 400

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

405 410 415

Gly Gly Gly Ser Gly Gly Ile Thr Cys Pro Pro Pro Met Ser Val Glu

420 425 430

His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg

435 440 445

Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu

450 455 460

Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr

465 470 475 480

Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Ser Lys Tyr

485 490 495

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

500 505 510

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

515 520 525

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

530 535 540

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

545 550 555 560

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

565 570 575

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

580 585 590

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

595 600 605

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

610 615 620

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

625 630 635 640

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

645 650 655

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

660 665 670

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys

675 680 685

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

690 695 700

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

705 710 715 720

Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp Lys

725 730

<210> 21

<211> 763

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

145 150 155 160

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

165 170 175

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

180 185 190

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

195 200 205

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

210 215 220

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

275 280 285

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

290 295 300

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

305 310 315 320

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

325 330 335

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

340 345 350

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

355 360 365

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

370 375 380

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val

405 410 415

Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu

420 425 430

Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

435 440 445

Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly

450 455 460

Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu

465 470 475 480

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp

485 490 495

Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

500 505 510

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu Ser

515 520 525

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

530 535 540

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

545 550 555 560

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

565 570 575

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

580 585 590

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

595 600 605

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

610 615 620

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

625 630 635 640

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

645 650 655

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

660 665 670

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

675 680 685

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

690 695 700

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

705 710 715 720

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

725 730 735

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

740 745 750

Leu Gly Gly Ser Ser His His His His His His

755 760

<210> 22

<211> 725

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

145 150 155 160

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

165 170 175

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

180 185 190

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

195 200 205

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

210 215 220

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro

275 280 285

Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr

290 295 300

Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly

305 310 315 320

Thr Cys Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala

325 330 335

His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Ser Gly Gly Ser Gly

340 345 350

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn

355 360 365

Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln

370 375 380

Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro

385 390 395 400

Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val

405 410 415

Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn

420 425 430

Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr

435 440 445

Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys

450 455 460

Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr

465 470 475 480

Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

485 490 495

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

500 505 510

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

515 520 525

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

530 535 540

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

545 550 555 560

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

565 570 575

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

580 585 590

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

595 600 605

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

610 615 620

Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

625 630 635 640

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

645 650 655

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

660 665 670

Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

675 680 685

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

690 695 700

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser Ser Asp Tyr Lys

705 710 715 720

Asp Asp Asp Asp Lys

725

<210> 23

<211> 763

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

145 150 155 160

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

165 170 175

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

180 185 190

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

195 200 205

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

210 215 220

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

275 280 285

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

290 295 300

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

305 310 315 320

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

325 330 335

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

340 345 350

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

355 360 365

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

370 375 380

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

385 390 395 400

Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu

405 410 415

Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp

420 425 430

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg

435 440 445

Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp

450 455 460

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln

465 470 475 480

Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg

485 490 495

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly

500 505 510

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ser

515 520 525

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

530 535 540

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

545 550 555 560

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

565 570 575

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

580 585 590

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

595 600 605

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

610 615 620

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

625 630 635 640

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

645 650 655

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

660 665 670

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

675 680 685

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

690 695 700

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

705 710 715 720

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

725 730 735

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

740 745 750

Leu Gly Gly Ser Ser His His His His His His

755 760

<210> 24

<211> 731

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

145 150 155 160

Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn

165 170 175

Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr

180 185 190

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

195 200 205

Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

225 230 235 240

Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Phe

245 250 255

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile

275 280 285

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

290 295 300

Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr

305 310 315 320

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Cys Glu Ser

325 330 335

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

340 345 350

Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys

355 360 365

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

370 375 380

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Ser Gly Gly Gly

385 390 395 400

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

405 410 415

Gly Gly Gly Ser Gly Gly Ile Thr Cys Pro Pro Pro Met Ser Val Glu

420 425 430

His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg

435 440 445

Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu

450 455 460

Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr

465 470 475 480

Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Ser Lys Tyr

485 490 495

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

500 505 510

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

515 520 525

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

530 535 540

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

545 550 555 560

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

565 570 575

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

580 585 590

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

595 600 605

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

610 615 620

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

625 630 635 640

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

645 650 655

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

660 665 670

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys

675 680 685

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

690 695 700

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

705 710 715 720

Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp Lys

725 730

<210> 25

<211> 763

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

145 150 155 160

Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn

165 170 175

Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr

180 185 190

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

195 200 205

Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

225 230 235 240

Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Phe

245 250 255

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

260 265 270

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

275 280 285

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

290 295 300

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

305 310 315 320

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

325 330 335

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

340 345 350

Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr

355 360 365

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

370 375 380

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Val

405 410 415

Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu

420 425 430

Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

435 440 445

Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly

450 455 460

Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu

465 470 475 480

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp

485 490 495

Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

500 505 510

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Ser Glu Ser

515 520 525

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

530 535 540

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

545 550 555 560

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

565 570 575

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

580 585 590

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

595 600 605

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

610 615 620

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

625 630 635 640

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

645 650 655

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

660 665 670

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

675 680 685

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

690 695 700

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

705 710 715 720

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

725 730 735

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

740 745 750

Leu Gly Gly Ser Ser His His His His His His

755 760

<210> 26

<211> 725

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

145 150 155 160

Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn

165 170 175

Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr

180 185 190

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

195 200 205

Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

225 230 235 240

Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Phe

245 250 255

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro

275 280 285

Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr

290 295 300

Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly

305 310 315 320

Thr Cys Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala

325 330 335

His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Ser Gly Gly Ser Gly

340 345 350

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn

355 360 365

Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln

370 375 380

Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro

385 390 395 400

Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val

405 410 415

Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn

420 425 430

Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr

435 440 445

Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys

450 455 460

Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr

465 470 475 480

Ser Gly Gly Gly Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

485 490 495

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

500 505 510

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

515 520 525

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

530 535 540

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

545 550 555 560

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

565 570 575

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

580 585 590

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

595 600 605

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

610 615 620

Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

625 630 635 640

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

645 650 655

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

660 665 670

Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

675 680 685

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

690 695 700

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Ser Ser Asp Tyr Lys

705 710 715 720

Asp Asp Asp Asp Lys

725

<210> 27

<211> 763

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

145 150 155 160

Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn

165 170 175

Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr

180 185 190

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

195 200 205

Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

225 230 235 240

Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Phe

245 250 255

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

260 265 270

Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

275 280 285

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

290 295 300

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

305 310 315 320

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

325 330 335

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

340 345 350

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

355 360 365

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

370 375 380

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

385 390 395 400

Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu

405 410 415

Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp

420 425 430

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg

435 440 445

Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp

450 455 460

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln

465 470 475 480

Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg

485 490 495

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly

500 505 510

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ser

515 520 525

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

530 535 540

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

545 550 555 560

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

565 570 575

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

580 585 590

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

595 600 605

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

610 615 620

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

625 630 635 640

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

645 650 655

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

660 665 670

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

675 680 685

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

690 695 700

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

705 710 715 720

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

725 730 735

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

740 745 750

Leu Gly Gly Ser Ser His His His His His His

755 760

<210> 28

<211> 721

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

145 150 155 160

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

165 170 175

Thr Ser Tyr Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

180 185 190

Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

195 200 205

Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

210 215 220

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Arg Leu Gly Phe Thr Thr Arg Asn Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile

275 280 285

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

290 295 300

Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr

305 310 315 320

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Cys Glu Ser

325 330 335

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

340 345 350

Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys

355 360 365

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

370 375 380

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser Ser Gly Gly Gly

385 390 395 400

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

405 410 415

Gly Gly Gly Ser Gly Gly Ile Thr Cys Pro Pro Pro Met Ser Val Glu

420 425 430

His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg

435 440 445

Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu

450 455 460

Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr

465 470 475 480

Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Ser Lys Tyr

485 490 495

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

500 505 510

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

515 520 525

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

530 535 540

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

545 550 555 560

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

565 570 575

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

580 585 590

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

595 600 605

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

610 615 620

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

625 630 635 640

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

645 650 655

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

660 665 670

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

675 680 685

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

690 695 700

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

705 710 715 720

Lys

<210> 29

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Thr Tyr Ala Met Asn

1 5

<210> 30

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys Asp

<210> 31

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

1 5 10

<210> 32

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala

1 5 10

<210> 33

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Gly Thr Asn Lys Arg Ala Pro

1 5

<210> 34

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 35

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Glu Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys Asp

<210> 36

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

His Ser Asn Phe Gly Asn Gly Tyr Val Ser Trp Phe Ala Tyr

1 5 10

<210> 37

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Gly Thr Asn Leu Arg Ala Pro

1 5

<210> 38

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Gly Thr Trp Leu Arg Ala Pro

1 5

<210> 39

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

Ala Leu Trp Arg Ser Gly Gly Trp Val

1 5

<210> 40

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

Ala Leu Trp Asn Lys Gly Gly Trp Val

1 5

<210> 41

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

Ala Leu Trp Asn Ser Gly Gly Trp Val

1 5

<210> 42

<211> 205

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ile Thr Cys Pro

130 135 140

Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser

145 150 155 160

Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys

165 170 175

Ala Gly Thr Cys Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn

180 185 190

Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

195 200 205

<210> 43

<211> 199

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly

65 70 75 80

Gly Gly Ser Leu Gln Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys

85 90 95

Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr

100 105 110

Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe

115 120 125

Leu Leu Glu Leu Gln Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile

130 135 140

His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser

145 150 155 160

Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu

165 170 175

Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val

180 185 190

Gln Met Phe Ile Asn Thr Ser

195

<210> 44

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Ser

115

<210> 45

<211> 65

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg

65

Claims

1. An anti-CD 3 antibody variant, said anti-CD 3 antibody comprising: a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises: VHCDR1, VHCDR2, VHCDR3; the light chain variable region comprises: VLCDR1, VLCDR2, VLCDR3; the amino acid sequence of the anti-CD 3 antibody comprises SEQ ID NO: VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of 29-34; the method is characterized in that: the mutation sites of the anti-CD 3 antibody variant comprise any 1 or more than 2 of the following sites:

the amino acid sequence of SEQ ID NO:30 in the sequence shown in the sequence table 30,

SEQ ID NO:31 in the sequence shown in the second and seventh sites,

SEQ ID NO:33 in the sequence shown in the sequence table 33,

SEQ ID NO:34, the fourth, fifth, sixth and seventh sites of the sequence shown in the specification.

2. The variant anti-CD 3 antibody of claim 1, wherein said mutation is an amino acid substitution.

3. The variant anti-CD 3 antibody of claim 1, wherein the mutation site of the variant comprises any 1 or 2 or more of the following sites:

SEQ ID NO:30 is substituted with E at the tenth position of A,

the amino acid sequence of SEQ ID NO:31 is replaced by S at the second position and G at the seventh position,

SEQ ID NO:33, the third position of the sequence is replaced by N and the fourth position of the sequence is replaced by K and L,

SEQ ID NO: 34Y at the fourth position is substituted by N or R, S at the fifth position is substituted by K, N at the sixth position is substituted by G, and L at the seventh position is substituted by G.

4. The anti-CD 3 antibody variant of claim 1,

the amino acid sequence of VHCDR2 is shown in SEQ ID NO. 35; alternatively, the first and second liquid crystal display panels may be,

the amino acid sequence of VHCDR3 is shown in SEQ ID NO: 36; alternatively, the first and second electrodes may be,

the amino acid sequence of VLCDR2 is shown in SEQ ID NO. 37 or 38; alternatively, the first and second liquid crystal display panels may be,

the amino acid sequence of VLCDR3 is shown in SEQ ID NO 39, 40 or 41.

5. The anti-CD 3 antibody variant of claim 1, wherein the amino acid sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 are as set forth in any one of the following sets of sequences:

(1)SEQ ID NO：29、35、31、32、37、39；

(2)SEQ ID NO：29、35、36、32、37、40；

(3)SEQ ID NO：29、35、36、32、38、41。

6. the anti-CD 3 antibody variant of claim 5, wherein the heavy chain variable region and/or the light chain variable region of said antibody variant is selected from the group consisting of SEQ ID NOs: 1.2 or 3, or at least 90% sequence identity thereto.

7. The anti-CD 3 antibody variant according to claim 1, wherein the heavy chain variable region and the light chain variable region have a mutation for disulfide bond formation at a position comprising any one or two or more of the following combinations, wherein the positions of the mutations are numbered according to EU, the heavy chain variable region is represented by VH, and the light chain variable region is represented by VL:

8. the anti-CD 3 antibody variant of claim 1, further comprising: a heavy chain constant region selected from human IgG1, igG2, igG3, or IgG4, or a variant thereof; and a light chain constant region selected from a human kappa, lambda chain or variant thereof; the heavy chain constant region comprises: an Fc fragment or variant thereof.

9. The anti-CD 3 antibody variant according to claim 1, wherein the antibody variant is an scFv comprising a heavy chain variable region, a light chain variable region, and a linker joining the heavy chain variable region and the light chain variable region, the amino acid sequence of the linker preferably being several GGGGS repeats, more preferably being 3 GGGGS repeats.

10. Use of an anti-CD 3 antibody variant according to any one of claims 1 to 9 in the manufacture of a medicament for the inhibition or treatment of cancer.

11. The use according to claim 10, wherein the cancer is selected from the following cancers or occurs at: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphatic cancer, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.

12. A nucleic acid molecule encoding the anti-CD 3 antibody variant of any one of claims 1 to 9.

13. An expression vector comprising the nucleic acid molecule of claim 12.

14. An anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule in heterodimeric form comprising a first monomer and a second monomer;

the first monomer includes: (a) an Fd fragment; (b) a light chain fragment and a first Fc chain;

the second monomer includes: (a) an Fd fragment; (b) a light chain fragment, an anti-CD 3 antibody fragment, and a second Fc chain;

the light chain fragment comprises a VL domain and a CL domain; the Fd fragment comprises a VH domain and a CH1 domain; in the first monomer or the second monomer, the light chain fragment pairs with the Fd fragment to form an anti-TAA Fab domain; wherein the light chain fragment of the first monomer is fused to the first Fc chain; the N-terminus of the anti-CD 3 antibody fragment is fused to the light chain fragment of the second monomer and the C-terminus is fused to the second Fc chain;

alternatively, the first and second liquid crystal display panels may be,

the first monomer includes: (a) an Fd fragment; (b) a light chain fragment, a cytokine domain, a first Fc chain;

the cytokine functional region comprises IL-15 and IL-15Ra; wherein the cytokine functional region is fused to the light chain fragment of the first monomer at the N-terminus and to the first Fc chain at the C-terminus; the N-terminus of the anti-CD 3 antibody fragment is fused to the light chain fragment of the second monomer and the C-terminus is fused to the second Fc chain;

alternatively, the first and second electrodes may be,

the first monomer includes: an anti-TAA antibody fragment, a cytokine functional region, a first Fc chain;

the second monomer includes: an anti-TAA antibody fragment, an anti-CD 3 antibody fragment, and a second Fc chain; wherein, the first and the second end of the pipe are connected with each other,

the N-terminus of the cytokine domain is fused to the anti-TAA antibody fragment of the first monomer and the C-terminus is fused to the first Fc chain; the N-terminus of the anti-CD 3 antibody fragment is fused to the anti-TAA antibody fragment of the second monomer and the C-terminus is fused to the second Fc chain;

the first Fc chain is interchangeable with the second Fc chain.

15. The anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14,

the anti-TAA antibody fragment is in the form of an scFv comprising: a heavy chain variable region (VH), a light chain variable region (VL), and a linker linking the heavy chain variable region and the light chain variable region; the amino acid fusion sequence of the anti-TAA antibody fragment is "VH to VL", or "VL to VH", from the N-terminus to the C-terminus of the peptide chain; "-" indicates a connecting fragment; alternatively, the first and second liquid crystal display panels may be,

the anti-CD 3 antibody fragment is in scFv format comprising: a heavy chain variable region (VH), a light chain variable region (VL), and a linker linking the heavy chain variable region and the light chain variable region; the fusion sequence of the amino acids of the anti-CD 3 antibody fragment is "VH to VL", or "VL to VH", from the N-terminus to the C-terminus of the peptide chain; "-" indicates a connecting fragment; alternatively, the first and second liquid crystal display panels may be,

the fusion sequence of the amino acid fragments of the cytokine functional region from the N-terminal to the C-terminal of the peptide chain is 'IL-15 to IL-15 Ra', or 'IL-15 Ra to IL-15'; "-" indicates a connecting fragment; preferably, the sequences of the IL-15 to IL-15Ra are shown in SEQ ID NO:42, and the sequences of the IL-15Ra to IL-15 are shown in SEQ ID NO: 43.

16. The anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14, wherein the amino acid sequence of the anti-CD 3 antibody fragment is selected from the group consisting of a specific antibody that binds CD3, an antibody fragment, a single domain antibody or a humanized form thereof; preferably, the amino acid sequence of the anti-CD 3 antibody fragment is selected from SP34, OKT3, UCTH1 or derivatives thereof.

17. The anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14, wherein the anti-CD 3 antibody fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region of the anti-CD 3 antibody fragment comprises: VHCDR1, VHCDR2, VHCDR3; the light chain variable region of the anti-CD 3 antibody fragment comprises: VLCDR1, VLCDR2, VLCDR3; the amino acid sequences of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are represented by any one of the sets of sequences in the anti-CD 3 antibody variant of claim 5.

18. The anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14, wherein IL-15Ra and IL-15 form an IL-15/IL-15Ra complex; the IL-15 includes: IL-15 and its mutations, truncations and various derivatives capable of binding IL-15Ra; IL-15Ra includes: IL-15Ra and mutations, truncations and various derivatives thereof capable of binding IL-15; preferably, the IL-15/IL-15Ra complex includes, but is not limited to, the mutation pattern shown in any combination of the following, counting from the first amino acid of the amino acid sequence of IL-15 or IL-15Ra to position 1; preferably, the parent sequence of the IL-15 is shown as SEQ ID NO: 44; the parent sequence of the IL-15Ra is shown as SEQ ID NO: 45:

。

19. The anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14, wherein the IL-15 includes but is not limited to the mutation pattern shown by any combination of the following, counted from the first amino acid of the amino acid sequence of IL-15 to position 1; preferably, the parent sequence of the IL-15 is shown in SEQ ID NO: 44:

。

20. The anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14, wherein the TAA is selected from the group consisting of CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276, PD-1, CEA, epcam, trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR VIII, C-MET, HER2, FGFR3, PSMA, PSCA, ephA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, and Glypican-3.

21. The anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14, wherein the first Fc chain and the second Fc chain are polymerized to form an Fc fragment; the Fc segment is selected from Human IgG1Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc or variants thereof, preferably from IgG1Fc or Human IgG4 Fc or variants thereof; the protein molecule is in the form of an Fc heterodimer; preferably, the Fc heterodimer comprises, but is not limited to, a combination of the following mutations, counted according to EU:

22. the anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14, wherein said protein molecule comprises an Fc fragment, which selects against immune effector functions, including but not limited to the following combinations of mutations, as counted according to EU:

23. the anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule according to claim 14, which is obtained by fusing amino acid fragments represented by any one of the following sequences:

(1)SEQ ID NO:05、SEQ ID NO:06、SEQ ID NO:07；

(2)SEQ ID NO:08、SEQ ID NO:06、SEQ ID NO:07；

(3)SEQ ID NO:09、SEQ ID NO:06、SEQ ID NO:07；

(4)SEQ ID NO:10、SEQ ID NO:06、SEQ ID NO:07；

(5)SEQ ID NO:05、SEQ ID NO:17、SEQ ID NO:07；

(6)SEQ ID NO:01、SEQ ID NO:18、SEQ ID NO:07；

(7)SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:07；

(8)SEQ ID NO:19、SEQ ID NO:18、SEQ ID NO:07；

(9)SEQ ID NO:20、SEQ ID NO:21；

(10)SEQ ID NO:22、SEQ ID NO:21；

(11)SEQ ID NO:20、SEQ ID NO:23；

(12)SEQ ID NO:22、SEQ ID NO:23；

(13)SEQ ID NO:24、SEQ ID NO:25；

(14)SEQ ID NO:26、SEQ ID NO:25；

(15)SEQ ID NO:24、SEQ ID NO:27；

(16)SEQ ID NO:26、SEQ ID NO:27。

24. a nucleic acid molecule encoding the anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule of any one of claims 14 to 23.

25. Use of the anti-Tumor Associated Antigen (TAA)/anti-CD 3 protein molecule of any one of claims 14 to 23 in the preparation of a medicament for inhibiting or treating cancer, including or occurring at a site selected from the group consisting of: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphatic cancer, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.

26. An anti-Tumor Associated Antigen (TAA) and IL-15/IL-15 Ra-containing fusion protein, comprising two polypeptide chains, wherein any one polypeptide chain comprises: anti-TAA antibody fragments, cytokine domains, fc fragments; the anti-TAA antibody fragment is in scFv format; the cytokine functional region comprises IL-15 and IL-15Ra; the N-terminus of the cytokine domain is fused to the anti-TAA antibody fragment and the C-terminus is fused to the Fc fragment.

27. The anti-Tumor Associated Antigen (TAA) and IL-15/IL-15 Ra-containing fusion protein according to claim 26, wherein IL-15Ra and IL-15 form an IL-15/IL-15Ra complex; IL-15 includes: IL-15 and mutations, truncations and various derivatives thereof that bind IL-15Ra; IL-15Ra includes: IL-15Ra and mutations, truncations and various derivatives thereof capable of binding IL-15; preferably, the IL-15/IL-15Ra complex includes, but is not limited to, the mutation patterns shown in any combination of the following, counted as 1 st amino acid from the first amino acid of the amino acid sequence of IL-15 or IL-15Ra; preferably, the parent sequence of the IL-15 is shown as SEQ ID NO: 44; the parent sequence of the IL-15Ra is shown as SEQ ID NO: 45:

。

28. The anti-Tumor Associated Antigen (TAA) and IL-15/IL-15 Ra-containing fusion protein of claim 26, wherein the IL-15 comprises, but is not limited to, any of the following mutations shown in any combination as numbered from the first amino acid of the amino acid sequence of IL-15 to position 1; preferably, the parent sequence of the IL-15 is shown in SEQ ID NO: 44:

。

29. The anti-Tumor Associated Antigen (TAA) and IL-15/IL-15 Ra-containing fusion protein of claim 26, wherein the fusion protein comprises an Fc fragment that selects against immune effector functions, including but not limited to the following combinations of mutations, as counted according to EU: :

30. the anti-Tumor Associated Antigen (TAA) and IL-15/IL-15 Ra-containing fusion protein of claim 26, wherein any one of the polypeptide chains of the fusion protein has an amino acid sequence as set forth in SEQ ID NO 28 or at least 90% sequence identity thereto.

31. A nucleic acid molecule encoding the anti-Tumor Associated Antigen (TAA) of any one of claims 26 to 30 and a fusion protein comprising IL-15/IL-15 Ra.

32. Use of an anti-Tumor Associated Antigen (TAA) and a fusion protein comprising IL-15/IL-15Ra according to any one of claims 26 to 30 in the manufacture of a medicament for the inhibition or treatment of cancer, including or occurring at a site selected from: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, bone marrow cancer, lymphatic cancer, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes.